7,8-bicycloakyl-chroman derivatives

ABSTRACT

7,8-Bicyclic-chroman derivatives of Formula I: 
     
       
         
         
             
             
         
       
     
     wherein the substituents are defined as in the specification or the pharmaceutically acceptable salts thereof, are disclosed. They are useful for the treatment of inflammatory disorders, neurodegenerative disorders and/or mitochondrial disorders. They are also useful in the manufacture of pharmaceutical formulations for the treatment of such conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. patent application claims priority under 35 U.S.C. 119(e) toU.S. Provisional Application Ser. No. 60/504,391, filed Sep. 19, 2003,and Ser. No. 60/541,737, filed Feb. 4, 2004 incorporated herein byreference in their entirety.

BACKGROUND INFORMATION

The present invention relates to novel 7,8-bicycloalkyl-chromanderivatives, pharmaceutical formulations containing them, and their usesas therapeutic agents, and syntheses therefor.

Cerebral ischemia or stroke refers to the severe diminution or cessationof blood flow to all or part of the brain. Cerebral ischemia can occuras a result of a number of causes or insults, including, but not limitedto cerebrovascular occlusion, thromboembolytic insult, cardiac failureand hemorrhagic accident. It is now known that pharmacologicintervention, if provided within a reasonable interval of the initialinsult, can significantly reduce cerebral tissue death followingcerebral ischemia.

The use of certain chroman-ylmethylamino derivatives for the treatmentof Parkinson's disease and epilepsy has been disclosed in U.S. Pat. Nos.5,663,294; 5,541,199; 5,670,667; 5,684,039; 5,756,521; 6,235,774; and6,331,561. The use of chromans for treating mitochondria associateddiseases including Alzheimer's disease, diabetes mellitus, Parkinson'sdisease, neuronal and cardiac ischemia, Huntington's disease, and strokeis disclosed in U.S. Pat. Nos. 6,498,191 and 6,511,966 and US patentapplication US 2003/0176448. Triphenyl phosphonium tocopherol analogshaving cardioprotective or mitochondrially targeted antioxidantproperties have been described by Gisar, J M in EP 545,283 and byMurphy, M. in Annals of the New York Academy of Sciences (2002), 959,263-274 and in U.S. Pat. No. 6,331,532, US 2202/00523242 and US2003/0069208. These applications neither teach nor suggest the7,8-bicycloalkyl-chromans derivatives described in this invention.

The use of antioxidants targeted to mitochondria shown to be effectiveat slowing disease progression has been reported by Jauslin, M L inFASEB Journal, express article 10.1096/fj.03-0240fje. Therapeuticbenefit of administering γ-tocopherol derivatives and metabolites asantioxidants and nitrogen oxide scavengers which treat high bloodpressure, thromboembolic diseases, cardiovascular disease, cancer,natriuretic disease, formation of neuropathological lesion and reducedimmune system response are disclosed in U.S. Pat. Nos. 6,555,575;6,242,479; 6,150,402; and 6,410,589. The use of certain chromanderivatives in cosmetic and dermatological preparations is disclosed inUS 2002/0127252. Beneficial effects of Vitamin E in the progression of anumber of major degenerative diseases of the nervous system is examinedin Fryer, Nutritional Neuroscience, (1998) Vol. 1, 327-351. Reduction ofthe inflammation marker CRP with 6-hydroxy chromans and with tocopherolshas been disclosed in commonly owned U.S. patent applications 60/426,764and US 2003/0100603, but these applications neither teach nor suggestthe use of 7,8-bicycloalkyl-chromans derivatives described in thisinvention.

The present invention addresses the desire to provide new therapies forconditions that affect millions of people, and particularly forconditions characterized by neuroprotection, neuroinflammation,cognitive disorders, neurodegenerative and/or mitochondrial dysfunctionconditions with novel 7,8-bicyloalkyl-chroman derivatives of Formula I.There remains a need for providing protection with agents that areeffective even if first administered after a significant time (e.g.about 5 hours) following an ischemic or oxidative insult. The compoundsof the present invention address this need.

SUMMARY OF THE INVENTION

The present invention is concerned with novel 7,8-bicyclo-chromanderivatives which may be useful in the manufacture of pharmaceuticalcompositions for treating or ameliorating a number of conditionscharacterized by neurodegeneration, oxidative stress or mitochondrialdisorders.

It has surprisingly been found that certain compounds of this inventionmay limit or prevent damage to organelles, cells, and tissues caused bymitochondrial dysfunction, oxidative stress or neuroinflammation, asdemonstrated by providing protection in standard experimental models ofmitochondrial dysfunction caused by MPP⁺ and the MPTP(1-methyl-4-phenylpyridinium or1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine) models or of oxidativestress caused by beta amyloid or high glutamate. These compounds havealso shown protection in the experimental model using FRDA fibroblasts,and in the MCAO animal model of cerebral ischemia showing potentneuroprotective protection with anti-inflammatory and anti-edemaactivity. Certain compounds may also show lipoxygenase inhibition.

Certain compounds of the present invention may also be useful in thetreatment or amelioration of indications characterized by oxidativestress and/or inflammation, including, but not limited to, diabetes,cardiopulmonary inflammatory disorders, skin inflammation or other skindisorders, premenstrual disease (PMS), chronic heart failure, rheumatoidarthritis, and muscle fatigue.

In a first aspect, the present invention concerns the novel compoundsrepresented by Formula I:

wherein:

-   -A-B- is —CH₂—(CH₂)₀₋₂—; —CH═CH—; —CH₂—O—; —CH₂—S—; or —CH₂—N—;-   n is 0 to 5;-   V is C₇₋₁₂-bicyclo[a.b.c]alkyl; C₇₋₁₂-bicyclo[a.b.c]alkenyl;    C₇₋₁₂-heterobicyclo[a.b.c]alkyl; or    C₇₋₁₂-heterobicyclo[a.b.c]alkenyl; and a, b, and c are 0 to 6; and    wherein the bicyclo ring is optionally substituted with one or more    substituents selected from C₁₋₆— alkyl, halogen, haloalkyl, carboxy,    alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo;-   R¹ is C₁₋₆ alkyl;-   R² is C₁₋₂₀ alkyl; optionally substituted C₂₋₂₀ alkenyl; halogen;    hydroxy; alkoxy; acyl; —C(O)OR; —S(O)₂OR; —NR′R″; —NH—C(═NH₂)—NR′R″;    —N—SO₂R; —NHC(O)NR′R″; —N(OH)C(O)NR′R″; —SO₂NR′R″; —C(O)NR′R″;    —S(O)₀₋₂R′″; —PO(OR)₂; triphenylphosphonium; trialkylphosphonium;    optionally substituted aryl; optionally substituted heterocyclyl;-   R³ is hydrogen; optionally substituted C₁₋₂₀ alkyl; C₂₋₂₀ alkenyl;    hydroxyalkyl; acyl; glucoside; phosphoryl; phosphoryloxyalkyl;    carboxyalkylcarbonyl; aminoalkylcarbonyl; or alkylketocarbonyl;-   R⁴ is hydrogen; halogen; nitro; cyano; optionally substituted alkyl;    aryl, aralkyl, heterocyclyl or heterocyclylalkyl, all optionally    substituted with alkyl, hydroxy, alkoxy, nitro, acyl, amino, oxo, or    —C(O)OR; optionally substituted alkenyl; hydroxy; alkoxy; nitro;    —C(O)OR; —C(O)NR′R″; —NR′R″; —NHC(O)NR′R″; —NR′—SO₂—R;    —NH—C(═NH₂)—NR′R″; —SO₂NR′R″, or —P(O)(OR)₂; or-   R³ and R⁴ taken together with the atoms to which they are attached    may form a heterocyclic ring;-   R is hydrogen; optionally substituted alkyl; optionally substituted    aryl; optionally substituted arylalkyl; optionally substituted    cycloalkyl; or optionally substituted heterocyclyl;-   R′ and R″ are independently of each other hydrogen; C₁₋₆ alkyl;    hydroxyalkyl; aminoalkyl; optionally substituted aryl; or optionally    substituted benzyl; or R′ and R″ taken together with the atom to    which they are attached may form a 5 to 8 membered aromatic,    saturated or unsaturated ring, optionally incorporating one    additional atom chosen from N, O, or S and optionally substituted    with a substituent selected from the group consisting of C₁₋₆ alkyl,    halo, cyano, alkylthio, lower alkoxy, phenyl, benzyl and carboxy;    and-   R′″ is optionally substituted C₁₋₆ alkyl; optionally substituted    aryl; or optionally substituted heterocyclyl; or    single stereoisomers and mixtures of stereoisomers, or the    pharmaceutically acceptable salts thereof.

In one embodiment, R³ is hydrogen. In another embodiment, R⁴ ishydrogen, C₁₋₆ alkyl optionally substituted with halogen, haloalkyl,hydroxy, alkoxy, amino, sulfanyl, carboxy, nitro or cyano; or C₂₋₁₂alkenyl optionally substituted with halogen, haloalkyl, hydroxy, alkoxy,amino, sulfanyl, carboxy, nitro or cyano.

In another embodiment, V is a bicyclo[2.2.1]heptane ring and thecompound is represented by Formula Ia:

wherein -A-B- is —CH₂—CH₂— or —CH═CH—; R¹, R², R³, R⁴, and n are asdefined in Formula I; m is in each occurrence independently 0-3; and R⁵is selected from optionally substituted C₁₋₆-alkyl, halogen, haloalkyl,carboxy, alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo.Preferably, V is a bicyclo[2.2.1]heptane ring and R² is C₁₋₆ alkyl;halogen; hydroxy; alkoxy; —C(O)OR; —SO₂NR′R″; —C(O)NR′R″; —SR′″;—PO(OR)₂; triphenylphosphonium; trialkylphosphonium; phenyl optionallysubstituted with C₁₋₆ alkyl, halogen, haloalkyl, carboxy,alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo; or heterocyclylselected from morpholine, piperidine, piperazine, thiazole,thiazolidine, isothiazole, oxazole, isoxazole, pyrazole, pyrazolidine,pyrazoline, imidazole, imidazolidine, benzothiazole, pyridine, pyrazine,pyrimidine, pyridazine, pyrrole, pyrrolidine, quinoline, quinazoline,purine, carbazole, benzimidazole, pyrimidine, thiophene, benzothiophene,pyran, tetrahydropyran, benzopyran, furan, tetrahydrofuran, indole,indoline, indazole, xanthene, thioxanthene, acridine, and quinuclidine,optionally substituted with C₁₋₆ alkyl, halogen, haloalkyl, carboxy,alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo; and morepreferably, V is a bicyclo[2.2.1]heptane ring, R² is —COOR and R ishydrogen or C₁₋₆ alkyl. In another embodiment V is abicyclo[2.2.1]heptane ring, R¹ and R² are C₁₋₆ alkyl and m and n are 0.

In another embodiment, V is a bicyclo[2.2.2]octane ring and the compoundis represented by Formula Ib:

wherein -A-B- is —CH₂—CH₂— or —CH═CH—; R¹, R², R³, R⁴, and n are asdefined in Formula I; m is in each occurrence independently 0-3; and R⁵is selected from optionally substituted C₁₋₆-alkyl, halogen, haloalkyl,carboxy, alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo.Preferably V is a bicyclo[2.2.2]octane ring and R² is C₁₋₆ alkyl;halogen; hydroxy; alkoxy; —C(O)OR; —SO₂NR′R″; —C(O)NR′R″; —SR′″;—PO(OR)₂; triphenylphosphonium; trialkylphosphonium; phenyl optionallysubstituted with C₁₋₆ alkyl, halogen, haloalkyl, carboxy,alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo; or heterocyclylselected from morpholine, piperidine, piperazine, thiazole,thiazolidine, isothiazole, oxazole, isoxazole, pyrazole, pyrazolidine,pyrazoline, imidazole, imidazolidine, benzothiazole, pyridine, pyrazine,pyrimidine, pyridazine, pyrrole, pyrrolidine, quinoline, quinazoline,purine, carbazole, benzimidazole, pyrimidine, thiophene, benzothiophene,pyran, tetrahydropyran, benzopyran, furan, tetrahydrofuran, indole,indoline, indazole, xanthene, thioxanthene, acridine, and quinuclidine,optionally substituted with C₁₋₆ alkyl, halogen, haloalkyl, carboxy,alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo; and morepreferably V is a bicyclo[2.2.2]octane ring, R² is —COOR, and R ishydrogen or C₁₋₆ alkyl. In another embodiment V is abicyclo[2.2.2]octane ring and R¹ and R² are independently of each otherC₁₋₆ alkyl and m and n are 0.

In another embodiment, V is a bicyclo[3.2.2]nonane ring and the compoundis represented by Formula Ic:

wherein -A-B- is —CH₂—CH₂— or —CH═CH—; R¹, R², R³, R⁴, and n are asdefined in Formula I; m is in each occurrence independently 0-3; and R⁵is selected from optionally substituted C₁₋₆-alkyl, halogen, haloalkyl,carboxy, alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo.Preferably V is a bicyclo[3.2.2]nonane ring and R² is C₁₋₆ alkyl;halogen; hydroxy; alkoxy; —C(O)OR; —SO₂NR′R″; —C(O)NR′R″; —SR′″;—PO(OR)₂; triphenylphosphonium; trialkylphosphonium; phenyl optionallysubstituted with C₁₋₆ alkyl, halogen, haloalkyl, carboxy,alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo; or heterocyclylselected from morpholine, piperidine, piperazine, thiazole,thiazolidine, isothiazole, oxazole, isoxazole, pyrazole, pyrazolidine,pyrazoline, imidazole, imidazolidine, benzothiazole, pyridine, pyrazine,pyrimidine, pyridazine, pyrrole, pyrrolidine, quinoline, quinazoline,purine, carbazole, benzimidazole, pyrimidine, thiophene, benzothiophene,pyran, tetrahydropyran, benzopyran, furan, tetrahydrofuran, indole,indoline, indazole, xanthene, thioxanthene, acridine, and quinuclidine,optionally substituted with C₁₋₆ alkyl, halogen, haloalkyl, carboxy,alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo; and morepreferably V is a bicyclo[3.2.2]nonane ring, R² is —COOR, and R ishydrogen or C₁₋₆ alkyl. In another embodiment V is abicyclo[3.2.2]nonane ring, R¹ and R² are independently of each otherC₁₋₆ alkyl and n is 0.

In another embodiment, -A-B- is —CH₂—CH₂— and n is 2 or 3, and in apreferred embodiment, -A-B- is —CH₂—CH₂—; n is 2 or 3 and V is abicyclo[2.2.1]heptane ring, a bicyclo[2.2.2]octane ring, or abicyclo[3.2.2]nonane ring. In another embodiment, -A-B- is —CH₂═CH₂— andn is 2 or 3, and in a preferred embodiment -A-B- is —CH₂═CH₂— and n is 2or 3, and V is a bicyclo[2.2.1]heptane ring, a bicyclo[2.2.2]octanering, or a bicyclo[3.2.2]nonane ring.

In another embodiment -A-B- is —CH₂—CH₂—; n is 2 or 3; R² is —C(O)OR,and R is hydrogen or C₁₋₆ alkyl. In another embodiment, -A-B- is—CH₂═CH₂—, n is 2 or 3; R² is —C(O)OR, and R is hydrogen or C₁₋₆ alkyl.

In another embodiment R¹ and R² are independently of each other C₁₋₆alkyl and n is 0, particularly R¹ and R² are independently of each otherC₁₋₆ alkyl, n is 0, and -A-B- is —CH₂—CH₂—. In another embodiment, R¹and R² are independently of each other C₁₋₆ alkyl, n is 0, and -A-B- is—CH₂═CH₂—.

In another embodiment the invention relates to a pharmaceuticalcomposition comprising a compound of Formula I or stereoisomers,mixtures of stereoisomers or pharmaceutically acceptable salts thereof,admixed with a pharmaceutically acceptable excipient, preferably to apharmaceutical composition comprising one or more compounds selectedfrom the group represented by the structures:

wherein R is hydrogen or C₁₋₄ alkyl.

In another aspect, the invention relates to the method of treating orameliorating a subject suffering from neurodegenerative, oxidativestress and mitochondrial disorders comprising administering to saidsubject a therapeutically effective amount of a compound of Formula I,or stereoisomers, mixture of stereoisomers or pharmaceuticallyacceptable salts thereof. In another preferred embodiment the inventionrelates to methods of treating or ameliorating a subject from acondition selected from stroke, cerebral ischemia, retinal ischemia,post-surgical cognitive dysfunctions, peripheral neuropathy/neuropathicpain, spinal cord injury, head injury and surgical trauma.

In another embodiment, the invention relates to a method of treating orameliorating a subject suffering from mitochondrial disorders such asbut not limited to epilepsy, Parkinsonism or Parkinson's disease,Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and other motorneuron diseases, macular degeneration, mitochondrial myopathy,encephalopathy, lactacidosis, stroke (MELAS), myoclonic epilepsy withragged red fibers (MERFF), Friedreich's ataxia and cerebellar ataxias ina mammal by administering to a mammal in need of such treatment atherapeutically effective amount of a compound of Formula I, orstereoisomers, mixture of stereoisomers or pharmaceutically acceptablesalts thereof.

In another preferred embodiment the invention relates to methods oftreating or ameliorating a subject suffering from an oxidative stressdisorder with inflammatory or autoimmune components, especially treatinga subject suffering from disorders including but not limited todiabetes, renal disease, premenstrual syndrome, asthma, chronicobstructive pulmonary disease (COPD), rheumatoid arthritis,osteoarthritis, muscle fatigue, irritable bowel syndrome, inflammatorybowel disease (IBD), premenstrual syndrome (PMS), and intermittentclaudication. In another preferred embodiment the invention relates tomethods of treating or ameliorating a subject suffering fromdermatological conditions characterized by oxidative stress, including,but not limited to age-related skin damage; damage resulting frominsults to the skin such as harmful ultraviolet (UV) radiation,pollution, stress and fatigue; contact dermatitis; skin irritation; skinpigmentation; psoriasis; or acne.

In another embodiment the invention relates to a method of inhibiting alipoxygenase enzyme in a subject in need of such inhibition whichcomprises administering to said subject a therapeutically effectiveamount of a compound of Formula I, including stereoisomers, mixture ofstereoisomers and pharmaceutically acceptable salts thereof,particularly the said method comprises administering a compound ofFormula I to a mammal suffering from asthma, chronic obstructivedisease, arthritis, rheumatoid arthritis, osteoarthritis, allergicrhinitis, psoriasis, a diabetic condition or cardiovascular disease.

Particularly preferred are those methods of treatment or ameliorationand uses in the manufacture of pharmaceutical compositions therefor,wherein the compound of Formula I is selected from the preferredcompounds or stereoisomers, mixture of stereoisomers or pharmaceuticallyacceptable salts thereof, and especially from the compounds selectedfrom:

-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   [3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonic    acid dimethyl ester;-   [3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   4-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-butane-1-sulfonic    acid dimethylamide;-   2-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-[7-(2-Methoxycarbonyl-ethyl)-2,7-dimethyl-2,7,9,10,11,12-hexahydro-1,8-dioxa-0-12-methano-triphenylen-2-yl]-propionic    acid methyl ester;-   3-[6-Hydroxy-2-methyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl]-propionic    acid;-   2-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-1-morpholin-4-yl-propan-1-one;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-1-morpholin-4-yl-propan-1-one;-   2-Methyl-2-(3-piperidin-1-yl-propyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   1-{3-[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propylsulfanyl]-2-methyl-propionyl}-pyrrolidine-2-carboxylic    acid;-   2-[3-(Benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-[3-(2-Hydroxy-ethylamino)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-[3-(2-Dimethylamino-ethylamino)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,6:9,12-Dimethano-9,10,11,12-tetrahydro-2-methylnaphtho[1,2-b]oxocan-8-ol;-   2-(2-Chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(pyridine-4-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-isobutylsulfanyl-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiophen-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiazol-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2,5-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2-[3-(Benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid, sodium salt;-   3-[6-Hydroxy-2-methyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl]-propionic    acid;-   Sodium salt of    3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid benzyl ester;-   3-(5-Bromo-6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   5-Bromo-2-methyl-2-(3-piperidin-1-yl-propyl)-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   5-Methoxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-1-piperidin-1-yl-propan-1-one;-   N-(2-Dimethylamino-ethyl)-3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionamide;-   2-Methyl-2-(3-piperidin-1-yl-propyl)-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-1-morpholin-4-yl-propan-1-one;-   2-[3-(2-Dimethylamino-ethylamino)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(pyridine-4-sulfonyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   2,2-Dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   5-Bromo-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   2,2-Dimethyl-3,4,7,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dim    ethyl-5-(3-methyl-but-2-enyl)-7,8,9,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dim    ethyl-5-(3-methyl-but-2-enyl)-3,4,7,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2,7,7-Tetramethyl-9-12-ethano-2,3,4,5,6,7,9,10,11,12-decahydro-1,8-dioxa-triphenylene;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-yl    ester;-   Phosphoric acid    mono-(2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl)    ester, disodium salt;-   2,2-Dimethyl-7,8,9,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   Phosphoric acid    mono-(2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano    2H-benzo[h]chromen-6-yl) ester, disodium salt;-   2,2-Dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-ethano    2H-benzo[h]chromen-6-ol;-   2,2,5-Trimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano    2H-benzo[h]chromen-6-ol;-   6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromene-5-carbonitrile;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   5-Hydroxymethyl-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   5-Bromo-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-[2-(tetrahydro-pyran-4-ylidene)-ethyl]-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   5-(2-Cyclohexylidene-ethyl)-2,2-dimethyl-3,4,7,8,9,10-hexahydro-1,10-ethano-2H-benzo[h]chromen-6-ol;-   1-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-5-yl)-ethanone;-   4-(6-Acetoxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-2H-benzo[h]chromen-5-yl)-4-oxo-butyric    acid;-   2,2-Dimethyl-5-nitro-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-but-2-enyl)-7,8,9,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   5-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-thiazolidine-2,4-dione;-   5-Hydroxy-3-(6-hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-3H-benzofuran-2-one;-   Phosphoric acid dibenzyl ester    2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   Phosphoric acid dibenzyl ester    2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-yl    ester;-   10-Methoxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   1-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-5-yl)-ethanone;-   4-[4-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-3-methyl-5-oxo-4,5-dihydro-pyrazol-1-yl]-benzoic    acid;-   4-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-2-methyl-5-propyl-2,4-dihydro-pyrazol-3-one;-   (6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethyl)-1-hydroxyurea;-   5-(1-Hydroxy-ethyl)-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo-[h]chromen-6-ol;    and-   Dimethylamino-acetic acid    2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-yl    ester.    In some embodiments the methods of treatment and uses in the    manufacture of pharmaceutical compositions therefor, comprise a    compound of Formula I selected from the following compounds in Table    1.

TABLE 1

3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic acid methyl ester;

3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chroman-2-yl)-propionic acid;

2-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;

2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;

4-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-2-methyl-5-propyl-2,4-dihydro-pyrazol-3-one;

[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonic acid dimethyl ester;

[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonic acid;

3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic acid methyl ester;

2-Methyl-2-thiazol-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;

4-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-butane-1-sulfonic acid dimethylamide;

2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;

2-Methyl-2-thiazol-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;

3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic acid methyl ester;

2-(3-Chloro-ethyl-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;

2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;

2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;

2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol; and

2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol.

Particularly preferred are those methods of treatment and uses in themanufacture of pharmaceutical compositions therefor, wherein thecompound is selected from the preferred compounds.

Another aspect of this invention is the processes for preparingcompounds of Formula I, and is set forth in “Description of theInvention”.

Certain embodiments of the invention provide novel and preferredcombinations of substituent groups pendant from the formulae of thedifferent inventions

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined below.

It will be understood by those skilled in the art with respect to anygroup containing one or more substituents that such groups are notintended to introduce any substitution or substitution patterns that aresterically impractical and/or physically non-feasible.

The term “acyl” refers to the groups —C(O)—H, —C(O)-(optionallysubstituted alkyl), —C(O)-(optionally substituted cycloalkyl),—C(O)-(optionally substituted alkenyl), —C(O)-(optionally substitutedcycloalkenyl), —C(O)-(optionally substituted aryl), and—C(O)-(optionally substituted heterocyclyl). This term is exemplifiedwith groups as formyl, acetyl, 4-oxo-4-yl-butiric acid.

The term “alkenyl” refers to a monoradical branched or unbranched,unsaturated or polyunsaturated hydrocarbon chain, having from about 2 to20 carbon atoms, more preferably about 2 to 10 carbon atoms. This termis exemplified by groups such as ethenyl, but-2-enyl,3-methyl-but-2-enyl (also referred to as “prenyl”, octa-2,6-dienyl,3,7-dimethyl-octa-2,6-dienyl (also referred to as “geranyl”), and thelike.

The term “substituted alkenyl” refers to refers to an alkenyl group inwhich 1 or more (up to about 5, preferably up to about 3) hydrogen atomsis replaced by a substituent independently selected from the group: ═O,═S, acyl, acyloxy, optionally substituted alkoxy, optionally substitutedamino (wherein the amino group may be a cyclic amine), azido, carboxyl,(optionally substituted alkoxy)carbonyl, (optionally substitutedamino)carbonyl, cyano, optionally substituted cycloalkyl, optionallysubstituted cycloalkenyl, optionally substituted heteroaryl, halogen,hydroxyl, nitro, sulfamoyl, sulfanyl, sulfinyl, sulfonyl, and sulfonicacid. Preferred examples of substituted alkenyl are5-vinyl-thiazolidine-2,4-dione, 3-vinyl-3-H-benzofuran-2-one,3-methyl-4-vinyl-5-oxo-4,5-dihydropyrazol-1-yl-benzoic acid,tetrahydropyran-4-ylidene-ethyl,2-methyl-4-vinyl-5-propyl-2,4-dihydro-pyrazol-3-one andcyclohexylidene-ethyl.

The term “acyloxy” refers to the moiety —O-acyl, including, for example,—O—C(O)-alkyl.

The term “alkoxy” refers to the groups —O-alkyl, —O-alkenyl,—O-cycloalkyl, —O-cycloalkenyl, and —O-alkynyl. Preferred alkoxy groupsare —O-alkyl and include, by way of example, methoxy, ethoxy, n-propoxy,iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

The term “substituted alkoxy” refers to the groups —O-(substitutedalkyl), —O-(substituted alkenyl), —O-(substituted cycloalkyl),—O-(substituted cycloalkenyl), —O-(substituted alkynyl) and—O-(optionally substituted alkylene)-alkoxy.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from about 1 to 20 carbonatoms, more preferably about 1 to 10 carbon atoms, and even morepreferably about 1 to 6 carbon atoms. The term “alkyl” also means acombination of linear or branched and cyclic saturated hydrocarbonradical consisting solely of carbon and hydrogen atoms. This term isexemplified by groups such as methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl, and the like. The term“lower alkyl refers to a monoradical branched or unbranched saturatedhydrocarbon chain of 1 to 6 atoms.

The term “substituted alkyl” refers to an alkyl group in which 1 or more(up to about 5, preferably up to about 3) hydrogen atoms is replaced bya substituent independently selected from the group: ═O, ═S, acyl,acyloxy, optionally substituted alkoxy, optionally substituted amino(wherein the amino group may be a cyclic amine), azido, carboxyl,(optionally substituted alkoxy)carbonyl, (optionally substitutedamino)carbonyl, cyano, optionally substituted cycloalkyl, optionallysubstituted cycloalkenyl, halogen, hydroxyl, nitro, sulfamoyl, sulfanyl,sulfinyl, sulfonyl, and sulfonic acid. One of the preferred optionalsubstituents for alkyl is hydroxy, exemplified by hydroxyalkyl groups,such as 2-hydroxyethyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl,and the like; dihydroxyalkyl groups (glycols), such as2,3-dihydroxypropyl, 3,4-dihydroxybutyl, 2,4-dihydroxybutyl, and thelike; aminoalkyl groups such as dimethyl aminoalkyl, piperidinylalkyl,morpholinylalkyl, and those compounds known as polyethylene glycols,polypropylene glycols and polybutylene glycols, and the like. Anotherpreferred optional substituent for alkyl is sulfanyl exemplified byallylsulfanyl, carboxypropylsulfanyl,2-methyl-propionyl-pyrrolidine-2-carboxylic acid,5-methyl-1-H-benzimidazol-2-yl-sulfanyl, sulfoxyethylsulfanyl,4,6-dimethyl-pyrimidin-2-ylsulfanyl, 4 carboxy-benzyl-sulfanyl,isobutylsulfanyl, and the like. Other preferred optional substituentsfor alkyl are —N-hydroxyureidyl, —N-hydroxythioureidyl or,—N-hydroxyacetamide. Other preferred alkyl substituents are halogenexemplified by chloro and bromo, acyl exemplified by methylcarbonyl,alkoxy, and heterocyclyl exemplified by morpholino and piperidino.

The term “alkylene” refers to a diradical derived from the above-definedmonoradical, alkyl. This term is exemplified by groups such as methylene(—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers [e.g., —CH₂CH₂CH₂—and —CH(CH₃)CH₂—] and the like.

The term “substituted alkylene” refers to a diradical derived from theabove-defined monoradical, substituted alkyl. Examples of substitutedalkylenes are chloromethylene (—CH(Cl)—), aminoethylene (—CH(NH₂)CH₂—),methylaminoethylene (—CH(NHMe)CH₂—), 2-carboxypropylene isomers(—CH₂CH(CO₂H)CH₂—), ethoxyethylene (—CH₂CH₂O—CH₂CH₂—),ethyl(N-methyl)aminoethylene (—CH₂CH₂N(CH₃)CH₂CH₂—),1-ethoxy-2-(2-ethoxy-ethoxy)ethylene (—CH₂CH₂O—CH₂CH₂—OCH₂CH₂—OCH₂CH₂—),and the like.

The term “Alzheimer's disease” (“AD”) refers to a progressive disease ofthe human central nervous system. It is manifested by dementia in theelderly, by disorientation, loss of memory, difficulty with language,calculation, or visual-spatial skills, and by psychiatricmanifestations. It is associated with degenerating neurons in severalregions of the brain.

The term “amino” refers to the group —NH₂ as well as to the groups —NHRor —NRR where each R is independently selected from the group:optionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted alkenyl, optionally substituted cycloalkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heterocyclyl, acyl, optionally substituted alkoxy, carboxyand alkoxycarbonyl, and where —NRR may be a cyclic amine.

The term “amyotrophic lateral sclerosis” (ALS) is the name given to acomplex of disorders that compromise upper and lower motor neurons.Patients may present with progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, or a combination ofthese conditions. The term ALS includes all types of ALS and other motorneuron diseases of similar clinical presentation. Amyotrophic lateralsclerosis (“ALS”), is also known as Lou Gehrig's disease or Charcotsyndrome. (http://www.lougehrigsdisease.net).

The term “aromatic” refers to a cyclic or polycyclic moiety having aconjugated unsaturated (4n+2) π electron system (where n is a positiveinteger), sometimes referred to as a delocalized π electron system.

The term “aryl” refers to an aromatic cyclic hydrocarbon group of from 6to 20 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed (fused) rings (e.g., naphthyl or anthryl). Preferred arylsinclude phenyl, naphthyl and the like.

The term “substituted aryl” refers to an aryl group as defined above,which unless otherwise constrained by the definition for the arylsubstituent, is substituted with from 1 to 5 substituents, andpreferably 1 to 3 substituents, independently selected from the groupconsisting of: hydroxy, thiol, acyl, acyloxy, optionally substitutedalkenyl, optionally substituted alkoxy, optionally substituted alkyl(such as tri-halomethyl), optionally substituted alkynyl, optionallysubstituted amino, optionally substituted aryl, optionally substitutedaryloxy, azido, carboxyl, (optionally substituted alkoxy)carbonyl,(optionally substituted amino)carbonyl, cyano, optionally substitutedcycloalkyl, optionally substituted cycloalkenyl, halogen, optionallysubstituted heterocyclyl, optionally substituted heterocyclooxy,hydroxyl, nitro, sulfanyl, sulfinyl, sulfanyl, and sulfonic acid.Preferred aryl substituents include alkyl, alkenyl, alkoxy, halo, cyano,nitro, haloalkyl, carboxy, amino, amido, sulfonamido, and sulfinyl.

The term “carboxy” or “carboxyl” refers to the moiety “—C(O)OH”, whichis also illustrated as “—COOH”.

The term “cognitive disorders” refers to disorders generallycharacterized by symptoms of forgetfulness, confusion, memory loss,impairment in attention and memory, behavioral and relation disorders,abulia, lack of interest, affective disturbances, and/or, in some casespoor personal care. These symptoms may arise as a result of the generalaging process and/or from organic brain disease, cerebrovasculardisease, head injury, or developmental or genetic defects. Cognitivedisorders include Alzheimer's disease, senile dementia, anxiety,HIV-related dementia, diabetic neuropathies; depression; Parkinson'sdisease; drug dependency; substance abuse; consciousness disorders,sleeping disorders, disorders of the circadian rhythm, mood disorders,epilepsy; Down's syndrome; Huntington's chorea or disease;stress-related somatic disorders; Creutzfeldt-Jacob disease; disordersassociated with panic, phobia or stress.

The term “convulsion” is used herein as meaning the violent involuntarycontraction or repeated contractions of the voluntary muscles.

The term “cycloalkyl” refers to non-aromatic cyclic hydrocarbon groupsof having about 3 to 40 (preferably about 4 to 15) carbon atoms having asingle ring or multiple condensed or bridged rings. Such cycloalkylgroups include, by way of example, single ring structures such ascyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, ormultiple ring structures such as adamantanyl, and the like. The term“cycloalkyl” additionally encompasses spiro systems wherein thecycloalkyl ring has a carbon ring atom in common with another ring.

The term “substituted cycloalkyl” refers to a cycloalkyl groupsubstituted with from 1 to 5 substituents, and preferably 1 to 3substituents, independently selected from the group consisting of: ═O,═S, acyl, acyloxy, optionally substituted alkenyl, optionallysubstituted alkoxy, optionally substituted alkyl (such astri-halomethyl), optionally substituted alkynyl, optionally substitutedamino, optionally substituted aryl, optionally substituted aryloxy,azido, carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, sulfanyl, and sulfonic acid. A cycloalkyl ringsubstituted with an alkyl group is also referred as “alkylcycloalkyl”.

The term “epilepsy” is used herein in its broadest sense, for example,as inclusive of grand mal, petit mal, and psychic equivalent orpsychomotor attacks.

The term “Friedreich's ataxia” as used herein also includes otherataxias, and is also sometimes referred to as hereditary ataxia,familiar ataxia, or Friedreich's tabes. Friedreich's ataxia is anautosomal recessive multi-system degenerative disorder disease thatresults in progressive damage to the nervous system and causes symptomsranging from muscle weakness and speech problems to heart disease.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The terms “heterocycle”, “heterocyclic”, “heterocyclo”, and“heterocyclyl” refer to a monovalent, saturated, partially unsaturatedor unsaturated (aromatic), carbocyclic radical having one or more ringsincorporating one, two, three or four heteroatoms within the ring(chosen from nitrogen, oxygen, and/or sulfur). Preferred heterocyclesinclude morpholine, piperidine, piperazine, thiazole, thiazolidine,isothiazole, oxazole, isoxazole, pyrazole, pyrazolidine, pyrazoline,imidazole, imidazolidine, benzothiazole, pyridine, pyrazine, pyrimidine,pyridazine, pyrrole, pyrrolidine, quinoline, quinazoline, purine,carbazole, benzimidazole, pyrimidine, thiophene, benzothiophene, pyran,tetrahydropyran, benzopyran, furan, tetrahydrofuran, indole, indoline,indazole, xanthene, thioxanthene, acridine, quinuclidine, and the like.

The terms “substituted heterocycle”, “substituted heterocyclic”,“substituted heterocyclo” and “substituted heterocyclyl” refer to aheterocycle group as defined above, which unless otherwise constrainedby the definition for the heterocycle, is substituted with from 1 to 5substituents, and preferably 1 to 3 substituents, independently selectedfrom the group consisting of: hydroxy, thiol, acyl, acyloxy, optionallysubstituted alkenyl, optionally substituted alkoxy, optionallysubstituted alkyl (such as tri-halomethyl), optionally substitutedalkynyl, optionally substituted amino, optionally substituted aryl,optionally substituted aryloxy, azido, carboxyl, (optionally substitutedalkoxy)carbonyl, (optionally substituted amino)carbonyl, cyano,optionally substituted cycloalkyl, optionally substituted cycloalkenyl,halogen, optionally substituted heterocyclyl, optionally substitutedheterocyclooxy, hydroxyl, nitro, sulfanyl, sulfinyl, and sulfonic acid.Preferred substituted heterocycles include pyrrolidine 2-carboxylicacid, thiazolidine-2,4-dione and 3-methyl-5-oxo-4,5-dihydro-1H-pyrazol.

The term “inflammation”, “inflammatory conditions”, or “inflammationconditions” includes but is not limited to muscle fatigue,osteoarthritis, rheumatoid arthritis, inflammatory bowel syndrome ordisorder, skin inflammation, such as atopic dermatitis, contactdermatitis, allergic dermatitis, xerosis, eczema, rosacea, seborrhea,psoriasis, atherosclerosis, thermal and radiation burns, acne, oilyskin, wrinkles, excessive cellulite, excessive pore size, intrinsic skinaging, photo aging, photo damage, harmful UV damage, keratinizationabnormalities, irritation including retinoid induced irritation,hirsutism, alopecia, dyspigmentation, inflammation due to wounds,scarring or stretch marks, loss of elasticity, skin atrophy andgingivitis.

The term “ischemia” refers to deficiency of blood to an organ or tissuedue to functional constriction or actual obstruction of a blood vessel.Cerebral ischemia, also known as stroke, usually results from theinterruption or reduction of blood and oxygen to the blood vessels ofthe brain; more rarely this may be the result of a hemorrhage. Signs ofstroke include paralysis, slurred speech, general confusion, impairmentof gait, cortical sensory loss over toes, foot and leg, and urinaryincontinence, to name just a few. Many types of heart disease includingcardiac arrhythmias or diseases due to cardiac structural abnormalitiesmay produce cerebral emboli. Atrial fibrillation from any cause,including rheumatic valvular disease, may result in emboli beingproduced which can migrate into the arteries of the brain. Emboliformation and migration can occur as a result of atheroscleroticcardiovascular disease and myocardial infarction. Emboli formation isalso a definite risk for intracardiac surgery and prosthetic valvereplacement. Heart bypass surgery and angioplasty can result in theformation of microemboli which can migrate into the arteries of thebrain and cause a series of occlusions in a number of arteries,resulting in mental impairment. Cerebral embolism is also the principalcomplication in the transplant of artificial hearts. Furthermore, theoverall risk of stroke after any type of general surgery is 0.2 to 1percent. The vegetations of acute and subacute bacterial endocarditiscan give rise to emboli which can occlude a major intracranial artery.Populations at risk of ischemia include but are not limited to patientsscheduled for coronary arterial bypass graft surgery (CABG), patients atrisk for postoperative complications, patients with subarachnoidhemorrhage (SAH), patients with a first or second ischemic stroke,patients with acute ischemic stroke, patients undergoing cardiopulmonaryresuscitation (CPR), patients with temporary lobotomy, patients withdominant hemisphere resection, patients receiving prophylactic brainradiation, patients with closed head trauma with neurological loss,patients with microvascular multi-infarct dementia, patients withhomozygous and heterozygous MELAS (Mitochondrial myopathy,encephalopathy, lactacidosis, stroke); patients with Myoclonic Epilepsywith Ragged Red Fibers (MERFF); patients with atherosclerotic orprogressive supranuclear palsy disease, patients with symptomatic andasymptomatic Huntington's disease, patients with neonatal asphyxia,patients with meningitis or encephalitis, patients with post herpeticneuropathy, patients with intermittent claudication, patients withspinal cord injury, patients with Huntington's disease, AmyotrophicLateral Sclerosis (ALS) or Friedreich's ataxia, patients with diabeticneuropathy or patients with a disease associated with a hypercoagulablestate secondary to systemic disease, carcinoma, vasoconstriction(including reversible cerebral vasoconstriction, e.g. migraine, trauma,idiopathy), or venous conditions (including dehydration, pulmonaryembolism, pericranial infection, postpartum and postoperative states andsystem cancer).

The term “isomers” or “stereoisomers” relates to compounds that haveidentical molecular formulae but that differ in the arrangement of theiratoms in space. Stereoisomers that are not mirror images of one anotherare termed “diastereoisomers” and stereoisomers that arenon-superimposable mirror images are termed “enantiomers”, or sometimesoptical isomers. A carbon atom bonded to four non-identical substituentsis termed a “chiral center”. Certain compounds of the present inventionhave one or more chiral centers and therefore may exist as eitherindividual stereoisomers or as a mixture of stereoisomers. Thisinvention includes all possible stereoisomers as individualstereoisomers or as a mixture of stereoisomers.

A “lipoxygenase-mediated condition” or a “disorder mediated bylipoxygenases” refers to a condition, disorder or disease related to orotherwise associated with a lipoxygenase enzyme or the inhibitionthereof, including, by way of example and without limitation, diseasesinvolving apoptosis in cancer cells such as prostatic cancer, gastriccancer, colorectal or esophageal cancer and airways carcinoma; diseasesinvolving hypoxia, or anoxia such as atherosclerosis, myocardialinfarction, cardiovascular disease, heart failure (including chronic andcongestive heart failure), cerebral ischemia, retinal ischemia,myocardial ischemia, post surgical cognitive dysfunction and otherischemias; diseases involving inflammation, including diabetes, arterialinflammation, inflammatory bowel disease, renal disease, pre-menstrualsyndrome, asthma, allergic rhinitis, gout; cardiopulmonary inflammation,rheumatoid arthritis, osteoarthritis, muscle fatigue and disorders ofthe skin such as acne; disorders of the airways such as asthma, chronicbronchitis, human airway carcinomas, mucus hypersecretion, chronicobstructive pulmonary disease (COPD) and adult respiratory distresssyndrome; diseases involving neurodegeneration and neuroinflammationincluding Alzheimer's, dementia and Parkinson's disease; peripheralneuropathy including spinal chord injury, head injury and surgicaltrauma, and allograft tissue and organ transplant rejection; diseasesinvolving the autoimmune system such as psoriasis, eczema, rheumatoidarthritis, and diabetes; and disorders involving the bone loss or boneformation.

The term “macular degeneration” includes an ophthalmic (eye) conditioncharacterized by progressive destruction and dysfunction of the centralretina (macula), and includes diseases that are all characterized by aprogressive loss of central vision. This term comprises age-relatedmacular degeneration (AMD). Age-related macular degeneration (AMD) is acollection of clinically recognizable ocular findings including drusen,retinal pigment epithelial (RPE) disturbance, pigment clumping and/ordropout, RPE detachment, geographic atrophy, subretinalneovascularization and disciform scar. Not all these manifestations areneeded for AMD to be considered present.

The term “mitochondrial diseases or disorders” of which hundreds ofvarieties have been identified—can cause a complex variety of symptoms.These include muscle weakness, muscle cramps, seizures, food reflux,learning disabilities, deafness, short stature, paralysis of eyemuscles, diabetes, cardiac problems and stroke-like episodes, to name afew. The symptoms can range in severity from life-threatening to almostunnoticeable, sometimes taking both extremes in members of the samefamily. Because some people have specific subsets of these symptoms,clinical researchers have grouped those that occur together into“syndromes,” producing a bewildering array of descriptive acronyms suchas MELAS (mitochondrial encephalomyopathy with lactic acidosis andstroke-like episodes) or MERFF (myoclonus epilepsy with ragged redfibers). This term also includes disorders such as Kearns-Sayre syndrome(KSS), Leigh's syndrome, maternally inherited Leigh's syndrome (MILS),myogastrointestinal encephalomyopathy (MNGIE), Neuropathy, ataxia andretinitis pigmentosa (NARP), Progressive external opthalmoplegia (PEO),and Pearson syndrome.

Mitochondrial myopathy, encephalopathy, lactacidosis, stroke (“MELAS”)is a progressive neurodegenerative disorder. The typical presentation ofpatients with MELAS syndrome includes features that comprise the name ofthe disorder such as mitochondrial encephalomyopathy, lactic acidosis,and stroke like episodes. Other features, such as diabetes mellitus andhearing loss, clearly are part of the disorder. MELAS is characterizedby stroke-like episodes and a mitochondrial myopathy.

The term “neurodegenerative disorders” refers to disorders characterizedby a loss of neurons and may or may not include a neuroinflammatoryprocess. Neurodegenerative disorders include stroke, head trauma,cerebral hypoxia, spinal cord injury, senile dementia, Alzheimer'sdisease, amyotrophic lateral sclerosis (ALS) and other motor neurondiseases, cerebral amyloid angiopathy, HIV-related dementia, Parkinson'sdisease, Huntington's disease, prion diseases, myasthenia gravis, Down'ssyndrome, Creutzfeldt-Jakob disease, Friedreich's ataxia, Fergusson andCritchley's ataxia and other ataxias, Leber's hereditary opticneuropathy diabetic neuropathy, neuropathic pain, encephalitis,meningitis, and Duchenne's muscular dystrophy.

The term “neuroinflammation” or “neuroinflammatory diseases, disordersor conditions” refers to diseases, disorders or conditions characterizedby large numbers of reactive microglia in postmortem brain samples,indicative of an active inflammatory process (McGeer E. G. and McGeer P.L., “Neurodegeneration and the immune system”. Calne D. B., ed.Neurodegenerative Diseases, 1994:277-300). Neuroinflammation refers toinflammation which occurs in response to brain injury or autoimmunedisorders, and has been shown to cause destruction of healthy neuronaland/or cerebral tissue. Neuroinflammation relates to mechanismsimplicated in a broad range of acute and chronic neurodegenerativedisorders, including stroke, head trauma, cerebral amyloid angiopathy,HIV-related dementia, Huntington's disease, prion diseases, meningitis,myelin degradation, epilepsy, Down's syndrome, post-ischemic braininjury, encephalopathy, Parkinson's disease, senile dementia,Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosisand certain disorders involving the peripheral nervous system, such asmyasthenia gravis and Duchenne's muscular dystrophy.

The terms “Parkinson's”, “Parkinsonism” and “Parkinsonian syndrome”(“PD”) as used herein include not only Parkinson's disease but alsodrug-induced Parkinsonism and post-encephalitic Parkinsonism.Parkinson's disease is also known as paralysis agitans or shaking palsy.It is characterized by tremor, muscular rigidity and loss of posturalreflexes. The disease usually progresses slowly with intervals of 10 to20 years elapsing before the symptoms cause incapacity. Due to theirmimicry of effects of Parkinson's disease, treatment of animals withmethamphetamine or MPTP has been used to generate models for Parkinson'sdisease. These animal models have been used to evaluate the efficacy ofvarious therapies for Parkinson's disease.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The term “pharmaceutically acceptable salt” refers to salts which retainthe biological effectiveness and properties of the compounds of thisinvention and which are not biologically or otherwise undesirable. Insome cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto. Pharmaceutically acceptable baseaddition salts can be prepared from inorganic and organic bases. Saltsderived from inorganic bases, include by way of example only, sodium,potassium, lithium, ammonium, calcium and magnesium salts. Salts derivedfrom organic bases include, but are not limited to, salts of primary,secondary and tertiary amines, such as alkyl amines, dialkyl amines,trialkyl amines, substituted alkyl amines, di(substituted alkyl) amines,tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines,trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl)amines, tri(substituted alkenyl) amines, cycloalkyl amines,di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkylamines, disubstituted cycloalkyl amine, trisubstituted cycloalkylamines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl)amines, substituted cycloalkenyl amines, disubstituted cycloalkenylamine, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines,triaryl amines, heterocyclic amines, diheterocyclic amines,triheterocyclic amines, mixed di- and tri-amines where at least two ofthe substituents on the amine are different and are selected from thegroup consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heterocyclic, and the like. Also included are amineswhere the two or three substituents, together with the amino nitrogen,form a heterocyclic group.

Specific examples of suitable amines include, by way of example only,isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine,tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

The term “seizure” is used herein as meaning the physical afflictioncharacterized by transient clouding of consciousness, generallyassociated with a disturbance in the electrical activity of the cortexof the brain. Illustrative of such seizures are those associated withpetit mal epilepsy.

The term “sulfanyl” or “thiol” refers to the groups: —S-(optionallysubstituted alkyl), —S-(optionally substituted aryl), —S-(optionallysubstituted heterocyclyl). Preferred sulfanyl groups include, by way ofexample, allylsulfanyl (—SCHCH₂═CH₂), n-(iso-butylsulfanyl)(—SCH₂CH(CH₃)₂), 3-thiazol-2-ylsulfanyl, captopril,3-carboxy-2-methylpropylsulfanyl, and the like.

The term “sulfonic acid” refers to the group: —S(O₂)—OH.

The term “therapeutically effective amount” refers to that amount of acompound of this invention that is sufficient to effect treatment, asdefined below, when administered to a mammal in need of such treatment.The therapeutically effective amount will vary depending upon thesubject and disease condition being treated, the weight and age of thesubject, the severity of the disease condition, the particular compoundchosen, the dosing regimen to be followed, timing of administration, themanner of administration and the like, all of which can readily bedetermined by one of ordinary skill in the art.

The term “treatment” or “treating” means any treatment of a disease ordisorder in a mammal, including:

-   -   preventing or protecting against the disease or disorder, that        is, causing the clinical symptoms not to develop;    -   inhibiting the disease or disorder, that is, arresting or        suppressing the development of clinical symptoms; and/or    -   relieving the disease or disorder that is, causing the        regression of clinical symptoms.        It will be understood by those skilled in the art that in human        medicine, it is not always possible to distinguish between        “preventing” and “suppressing” since the ultimate inductive        event or events may be unknown, latent, or the patient is not        ascertained until well after the occurrence of the event or        events. Therefore, as used herein the term “prophylaxis” is        intended as an element of “treatment” to encompass both        “preventing” and “suppressing” as defined herein. The term        “protection,” as used herein, is meant to include “prophylaxis.”

Nomenclature

In general, the nomenclature used in this application was generated withthe help of version 2.2 of the AUTONOM™ naming package within theChemOffice® version 7.0.3 suite of programs by CambridgeSoft Corp(Cambridge, Mass.).

A compound of Formula I wherein V is bicyclo[2.2.1]heptane, -A-B- is—CH₂—CH₂—, n is 2, R¹ is methyl, R² is —C(O)OCH₃, R³ and R⁴ are hydrogenis named3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester.

Synthesis of the Compounds of the Invention Synthetic ReactionParameters

The terms “solvent”, “inert organic solvent” or “inert solvent” mean asolvent inert under the conditions of the reaction being described inconjunction therewith. Solvents employed in synthesis of the compoundsof the invention include, for example, methanol (“MeOH”), acetone,water, acetonitrile, 1,4-dioxane, dimethylformamide (“DMF”), benzene,toluene, tetrahydrofuran (“THF”), chloroform, methylene chloride (alsonamed dichloromethane ((“DCM”), diethyl ether, ethyl acetate (“EtOAc”),pyridine and the like, as well as mixtures thereof. Unless specified tothe contrary, the solvents used in the reactions of the presentinvention are inert organic solvents.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure within a temperature range from 0° C. to110° C. (preferably from 0° C. to 25° C.; most preferably at “room” or“ambient” temperature (“RT”), e.g., 20° C.). Further, unless otherwisespecified, the reaction times and conditions are intended to beapproximate, e.g., taking place at about atmospheric pressure within atemperature range of about 0° C. to about 110° C. (preferably from about0° C. to about 25° C.; most preferably at about “room” or “ambient”temperature, e.g., approximately 20° C.) over a period of about 1 toabout 10 hours (preferably about 5 hours).

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column on silica gel, thin-layer chromatography onsilica gel or thick-layer chromatography on silica gel, or a combinationof these procedures. Specific illustrations of suitable separation andisolation procedures can be had by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures canalso be used.

Referring to Reaction Scheme 1, ethyl 2-ketopentoate (Formula 101)undergoes a Grignard reaction with vinylmagnesium bromide to give afuranone of Formula 102. The hydroquinone derivative of Formula 103wherein R⁴ is hydrogen and V is a bicyclo[2.2.1]heptane ring used inStep 2 is available commercially from TCI, Portland, Oreg. or fromFisher Scientific, Somerville, N.J. Other hydroquinone derivatives ofFormula 103 can be prepared by methods known to those of ordinary skillin the art, by Diets Adler reaction of a quinone and a cyclohexadiene.Reaction of compound 103 with the vinyl furanone of Formula 102, in thepresence of boron trifluoride in an inert solvent such as dioxane givesthe acid of Formula 104. Esterification in Step 3 to the correspondingcompound of Formula 105 can be done under conditions well known in theart, such as with an alcohol in the presence of an acid such ashydrochloric acid. In Step 4, the hydroxyl group is protected with, forexample, chloromethyl methyl ether in the presence of a base such asdiisopropylethylamine in solvent such as methylene chloride, and theprotected ester of Formula 106 is reduced and deprotected to give thealcohol of Formula 107. The reducing agents that can be used includelithium aluminum hydride, lithium borohydride or sodium borohydride inan organic solvent such as tetrahydrofuran (THF) or diethylether.Deprotection can be done with an acid such as hydrochloric acid in asolvent such as methanol. In methods well known to the skilledpractitioner the alcohol derivative of Formula 107 can be convertedinto, for example, a halide of Formula 108, a sulfonate of Formula 109,a phosphonate of Formula 110, a thiol of Formula III, or a sulfonamideof Formula 112, as exemplified in the examples. In the reaction schemedescribed above, V, R, R′, R″ and R⁴ have the meanings defined supra.

Compounds of Formula I wherein the 5-position is substituted with asubstituted alkyl of at least two carbons or a substituted alkenyl, canbe prepared following Scheme 3. In Scheme 3, R^(3.1) and R^(3.2) arehydrocarbon groups, preferably unsubstituted alkyl groups, V has themeaning of a bicyclic ring as defined in Formula I. Pro is a protectivegroup and Z¹ and Z² are the substituents of interest for the alkyl groupat the 5 position, or Z¹ is hydrogen and Z² is the substituent ofinterest for the alkyl group.

The chroman of Formula 301 is brominated in an inert solvent to give themethylbromide derivative 302, which is then converted to the phosphoniumsalt of Formula 303 by addition of triphenylphosphine. The hydroxy groupof the phosphonium salt derivative 303, can be protected with forexample, the methoxymethyl (MOM) group by reaction withchloromethylmethyl ether to give a MOM-protected compound of Formula304. In the next step a Wittig reaction is performed with an aldehyde ora ketone of formula Z¹Z²C(O), in an inert solvent in the presence of astrong base, such as sodium alkoxide or sodium hydride, preferablysodium hydride to give a compound of Formula 305. Hydrogenation of thedouble bond of compound of Formula 305 in a hydrogen atmosphere in thepresence of a catalyst such as Palladium on charcoal can yield compoundof Formula 306, which after removal of the protective group can give thedesired saturated compound of Formula 308. Removal of the protectinggroup can be effected with an acid such as hydrochloric acid in asolvent such as an alcohol, preferably in methanol. Deprotection ofcompound of Formula 305 with an acid can give the unsaturated compoundof Formula 307, which if desired, can also be hydrogenated to give thecompound of Formula 308, under the conditions described herein.

Alternatively, the chromans of Formula 301, wherein R^(3.1) and R^(3.2)have respectively the meaning of R¹ and (CH₂)_(n)R² of Formula I asdefined supra, and further wherein V has the meaning of a bicyclic ringas defined in Formula I, can be brominated as described herein to give abromide derivative of Formula 302, which followed by the treatment witha compound of Formula Z³YH wherein Y is oxygen, sulfur or nitrogen andZ³ is the desired substituent, in the presence of a mild base such assodium or potassium carbonate, sodium or potassium bicarbonate, in aninert solvent, preferably methylene chloride, can give a compound ofFormula 309.

Preferred Compounds

The compounds of Formula I encompass the chroman derivatives of theinvention as disclosed, and/or the pharmaceutically acceptable salts ofsuch compounds. In addition, the compounds of this invention include theindividual stereochemical isomers and mixtures thereof, arising from theselection of substituent groups. For Example,3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid exists as 4 single stereoisomers:

and the chemical name3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid as used herein includes all 4 stereoisomers individually or asmixtures of stereoisomers. The mixture of stereoisomers can be separatedinto their individual stereoisomers by procedures well known in the artsuch as for example chiral chromatography.

It will be understood by those skilled in the art with respect to anygroup containing one or more substituents that such groups are notintended to introduce any substitution or substitution patterns that aresterically impractical and/or synthetically non-feasible.

Preferred for the compounds, pharmaceutical formulations, methods ofmanufacture and use of the present invention are the followingcombinations and permutations of substituent groups of Formula I.

Utility, Testing and Administration General Utility

Compound, compositions, formulations, and methods of the presentinvention can be used for the treatment of disorders characterized byneuroinflammation, neurodegeneration, defective mitochondrial activity,oxidative stress and inflammation. In particular, compounds of thepresent invention can be used in the treatment of stroke or ofneurodegeneration caused by stroke, particularly for the reduction ofedema in the brains of stroke patients. Compounds of the presentinvention can also be used in the treatment of diseases such asdegenerative diseases of the brain ((Wernicke-Korsakoff disease,Kreuzfeldt-Jakob disease (KJD), Hallervorden-Spatz disease, Schilder'sdisease, Alzheimer's disease, senile dementia, Down's syndrome in middleage, Abercrombie's disease, Prion diseases, Zellweger syndrome, Alper'sSyndrome), spinocerebellar degenerations (spinal ataxia, cerebellarcortical degenerations, Friedreich's ataxia and other ataxias), multiplesystem degenerations (Menzel, Dejerine-Thomas, Shy-Drager, and MachadoJoseph), systemic disorders (Refsum disease, ataxia telangiectasia),epilepsy, mitochondrial disorders (MELAS, MERFF, KSS, Leigh's, MI LS,MNGIE, NARP, PEO, Pearson), demyelinating core disorders (multiplesclerosis, acute transverse myelitis), muscular atrophies (amyotrophiclateral sclerosis (ALS), multiple sclerosis (MS), infantile spinalmuscular atrophy, Huntington's disease, spinobulbar atrophy (SBA),juvenile spinal muscular atrophy, myasthenia gravis and other motorneuron diseases), movement disorder (drug-induced Parkinsonism orParkinson's disease), retinopathy (Leber's hereditary optic neuropathy,age-related macular degeneration (AMD), cataracts), cerebral ischemia(“stroke” most often caused by thrombosis, vasoconstriction andembolism), myocardial ischemia (including chronic stable angina, anginapectoris, unstable angina and Prinzmetal's angina, silent ischemia,reinfarction, reocclusion, restenosis, myocardial infarction and otherforms of heart disease), diabetes, renal disease, pre-menstrual syndrome(PMS), asthma, cardiopulmonary inflammatory disorders, chronic heartfailure, rheumatoid arthritis, muscle fatigue, irritable bowel syndrome,inflammatory bowel disease, intermittent claudication and for thepreservation of allograft tissue for transplantation. Certain compoundsof the present invention can also be useful in treating conditionsfalling with the group of dermatologic conditions, in particularprevention and protecting skin tissue against age-related damage ordamage resulting from insults such as harmful ultraviolet (UV)radiation, stress and fatigue, and in the treatment of contactdermatitis, skin irritation, skin pigmentation, psoriasis, or acne.

Without subscribing to a particular theory or mechanism of action,compounds of the invention may target certain enzymes known as“oxidoreductases” that function widely across a variety of physiologicalprocesses; more particularly certain compounds of the present inventionmay target lipoxygenases such as 5-lipoxygenase, 15 lipoxygenase and/or12/15-lipoxygenase. In particular, oxidoreductases catalyze reactions inwhich two molecules interact so that one molecule is oxidized and theother is reduced. Abnormalities in oxidoreductase activity may underliesuch disorders as congestive heart failure, respiratory chain defects(e.g., abnormalities associated with enzymes of the respiratory chain,acute respiratory distress syndrome (ARDS)), glycogen storage disease,end-stage renal disease, and rheumatoid arthritis. Inhibitors oflipoxygenases are known to be useful in the prevention or treatment offor example, asthma, arthritis, chronic obstructive pulmonary disease(COPD), osteoarthritis, psoriasis, diabetes, ulcers, bone loss,atherosclerosis, and myocardial infarction.

Testing

This section describes how compositions incorporating compositions ofthe present invention are selected, using in vitro and/or in vivomodels, for example, and used as therapeutic interventions in theexemplary indications, i.e., stroke, epilepsy, Parkinson's disease,Friedreich's ataxia, MELAS, macular degeneration, ALS, and Alzheimer'sdisease.

MPTP/MPP⁺-induced neurodegeneration of dopaminergic neurons is a wellcharacterized model which is therefore widely used to understand thepathogenesis of Parkinson's disease. The compounds were tested againstMPTP/MPP⁺ induced neuronal death in vitro and in vivo as shown in thefollowing examples. In vitro evaluation of protection againstmitochondrial dysfunction is carried out using substantia nigra-deriveddopaminergic progenitor cell line (as described in Son J H, et al J W.(1999) J Neurosci, 19:10-20), exposed to 1-methyl-4-phenylpyridinium(MPP⁺). In vivo evaluation was carried out using mice that had beentreated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), aneurotoxin. MPTP is metabolized by astrocytes into1-methyl-phenylpyridinium (MPP⁺), a substrate for the dopaminetransporter which then selectively inhibits complex 1 of themitochondrial electron transport chain. This results in depletion ofATP, the production of reactive oxygen species and, consequently celldeath. In a number of species, including humans, non-primates androdents, MPTP produces an irreversible and severe parkinsonian syndromewhich includes virtually all the clinical features of the disease. Thestriking pathologic and clinical similarities between idiopathicParkinson's disease and MPTP-induced Parkinsonism suggest that the twodisorders share common pathogenic mechanism.

A cellular assay using FRDA-patient derived fibroblasts (as described byJauslin, M L et al, Human Molecular Genetics 11; 3055-3063 (2002)); wasused to determine the protective effects of the test compounds byanalyzing survival of dermal fibroblasts taken from FRDA patients andunaffected normal donors under conditions of partial GSH depletion.Exposure of FRDA fibroblasts to BSO (L-buthionine (S,R)-sulfoximine)under conditions of restricted selenium causes depletion of cellularglutathione (GSH) and severe plasma membrane damage leading to celldeath. Pre-incubation with the test compounds before the addition of BSOwas used to determine if they could protect FRDA cells from BSO-mediatedcell death.

In experiments carried out in support of the present invention accordingto methods detailed in the Examples, anoxia/ischemia was induced inprimary cultures of hippocampal neuronal cells, oxidative stress wasinduced in a neuronal cell line, and compounds were tested for theirability to prevent cell death. Using in vitro assays the potency andefficacy of test articles against redox injury and cell death can beestablished in a high throughput manner and the compounds found to haveactivity in those in vitro assays are then further tested in one or moreanimal models of cerebral ischemia (“stroke”), such as the middlecerebral artery occlusion (MCAO) model in rats.

Primary embryonic hippocampal neuronal cells are widely recognized asuseful in models of neuronal function. Briefly, primary cultures ofhippocampal neurons are used to test compounds for activity in neuronalprotection. Hippocampal cultures are typically prepared from 18- to19-day fetal rats. At this age, the generation of pyramidal neurons,which begins in the rat at about E15, is essentially complete. The braintissue at this stage is relatively easy to dissociate, the meninges areremoved readily, and the number of glial cells still is relativelymodest (Park L C, Calingasan N.Y., Uchida K, Zhang H, Gibson G E.(2000). Metabolic impairment elicits brain cell type-selective changesin oxidative stress and cell death in culture. J Neurochem74(1):114-124). In order to evaluate the activity of compounds of thepresent invention, a test compound is assessed for its ability toprotect cells against beta-amyloid induced oxidative stress inhippocampal neurons, as detailed in Examples.

Protection against redox stress can be further evaluated in cell cultureusing high glutamate induced oxidative stress (HGOS) in mousedopaminergic cell lines. The cytotoxic effect of glutamate is not due toexcitotoxicity, as this cell line is devoid of inotropic glutamatereceptors. Rather, the glutamate-induced toxicity of dopaminergic cellsis associated with an inhibition of cystine transport which subsequentlyleads to depletion of intracellular glutathione (GSH) levels (Murphy T.H., et al. Neuron 2, 1547-1558, 1989), activation of neuronal12-lipoxygenase (Li, Y. et al., Neuron 19, 453-463, 1997), increased ROSproduction (Tan S. et al., J. Cell Biol. 141, 1423-1432, 1998) andelevated intracellular Ca²⁺ (Li, Y. et al., see supra). Some moleculeswere measured for their ability to protect cells againstglutamate-induced stress and the assay is detailed in Examples.

Further validation of neuroantiinflammatory activity of compounds can beassessed in vitro by the inhibition of IL-1.beta. release from amicroglial cell line.

Interleukin-1 (IL-1) is a proinflammatory cytokine that exists in twoseparate forms that share 30% sequence homology (alpha and beta).Constitutive expression of IL-1 is low in the brain but levels of bothforms of this cytokine increase dramatically after injury. There issubstantial evidence that IL-1 is an important mediator ofneurodegeneration induced by cerebral ischemia (Touzani O et al, JNeuroimmunol., 100:203-215, (1999)). Both IL-1 forms are rapidly inducedin experimental models of stroke and administration of recombinant IL-1beta enhances ischemic injury (see Hill J K. et al. Brain Res.820:45-54, (1999), Hilhouse E W et al. Neurosci Lett 249:177-179,(1998), Loddick S A et al J Cereb Blood Flow Metab 16:932-940, (1996),Stroemer R P et al., J Cereb Blood Flow Metab. 18:833-839, (1998)).Conversely, blocking IL-1 actions with a receptor antagonist or aneutralizing antibody markedly reduces neuronal death and inflammationin models of ischemic damage (see Betz A L, J Cereb Blood Flow Metab15:547-551, (1995); Relton J K, Brain Res Bull 29:243-246, (1992);Yamasaki Y et al, Stroke 26:676-680, (1995)). Furthermore, mice withdecreased IL-1.beta. production (caspase-1 knockouts) are significantlyprotected from ischemic injury (Schielke G P, et al. J Cereb Blood FlowMetab 18:180-185, (1998)) and IL-1α and β double knockouts exhibitdramatically reduced ischemic infarct volumes compared with wild-typemice (87% reduction in cortex) (Boutin H et al., J Neurosci21:5528-5534, (2001)).

In addition to a role in ischemic damage, IL-1 elevation has beenassociated with many neurodegenerative diseases. There is increasingevidence for a role of IL-1 in Alzheimer's Disease (AD) (Mrak R E et al.Neurobiol Aging 22(6):903-908, (2001)). Elevated levels of IL-1β havebeen shown to surround amyloid plaques in the disease and recent geneticstudies have indicated that a polymorphism in IL-1α is linked to anincreased risk of AD (3-6 fold increase) (Griffin W S et al., J LeukocBiol 72(2):233-238, (2002)). This polymorphism has also been correlatedwith rate of cognitive decline in AD patients (Murphy G M et al.,Neurology, 56(11)1595-1597, (2001)). The risk of AD is increased evenfurther when the polymorphism in IL-1.alpha. is found in combinationwith another polymorphism in IL-1β (see Griffin W S, supra), providingconvincing evidence that these cytokines play an important role in thepathology of the disease.

This assay measures the release of IL-1β from a mouse microglial cellline following an inflammatory challenge with LPS and interferon-gamma.The ability of test articles to inhibit microglial cell activation andIL-1β release is determined by co-incubation of the test article withthe inflammatory challenge.

Cerebral ischemic insults are modeled in animals by occluding-vesselsto, or within, the cranium (Molinari, G. F., 1986, in H. J. M. Barnett,et al., (Eds) Stroke: Pathophysiology, Diagnosis and Management, Vol. 1,Churchill Livingstone, N.Y.). The rat middle cerebral artery occlusion(MCAO) model is one of the most widely used techniques to inducetransient focal cerebral ischemia approximating cerebral ischemic damagein humans, e.g., those who suffer from a stroke. The middle cerebralartery used as the ischemic trigger in this model is the most affectedvessel in human stroke. The model also entails a period of reperfusion,which typically occurs in human stroke victims. MCAO involving atwo-hour occlusion has been found to produce the maximum size ofcortical infarction obtainable without increased mortality attwenty-four hours.

Further validation of efficacy in neuroprotection can be assessed infunctional tests, such as the grip strength test or the rotorod test.Animals treated with compounds that show neuroprotection maintain theirpre-MCAO grip strength values after MCAO, as compared to untreatedanimals, which showed a significant reduction in grip strength,indicating loss of sensorimotor function. Likewise, animals treated withcompounds that show neuroprotection also maintained their pre-MCAOrotorod activity scores after MCAO, as compared to untreated animals,which showed a significant reduction in rotorod scores, indicating lossof sensorimotor function at higher brain levels.

In vitro cell-based assays for inflammation are well known in the art,for example e-selectin (also named Endothelial Leukocyte AdhesionMolecule or ELAM) or C-reactive protein (CRP). The ELAM assay measuresin vitro activity of the test compounds in reducing expression of ELAMin activated endothelial cells. Briefly, endothelial cells are createdby adding known activators such as lipopolysaccharides, TNF or IL-1,alone or in some combination. Activated cells produce ELAM, which can bemeasured using, for example, an E-selectin monoclonal antibody-basedELISA assay. Similarly the CRP assay measures in vitro activity of testcompounds in reducing expression of CRP in Human Hep3B epithelial cells.The activated cells produce CRP, which can be measured with a CRP ELISAassay.

In vivo evaluation of anti-inflammatory activity can be determined bywell characterized assays measuring Carrageenan-Induced Paw Edema and byMouse Ear Inflammatory Response to Topical Arachidonic Acid. (Gabor, M.,Mouse Ear Inflammation Models and their Pharmacological Applications,2000). Carrageenan-Induced Paw Edema is a model of inflammation, whichcauses time-dependent edema formation following carrageenanadministration into the intraplantar surface of a rat paw. Theapplication of arachidonic acid (AA) to the ears of mice producesimmediate vasodilation and erythema, followed by the abrupt developmentof edema, which is maximal at 40 to 60 min. The onset of edema coincideswith the extravasations of protein and leukocytes. After one hour theedema wanes rapidly and the inflammatory cells leave the tissue so thatat 6 hours the ears have returned to near normal. These assays, asdescribed in the Examples, measure a test compound's ability to treatthese inflammatory processes via systemic and topical routes ofadministration.

The 5-lipoxygenase pathway is a major synthetic pathway relevant tohuman inflammatory disease. 5-lipoxygenase catalyses the two first stepsin the oxygenation of arachidonic acid (a polyunsaturated 20-carbonfatty acid) to leukotrienes. Leukotrienes are known to be importantmediators of inflammatory and allergic reactions. The first step in thesynthesis of leukotrienes, which is catalyzed by 5-lipoxygenase, is theformation of 5-HPETE. The rearrangement of 5-HPETE to form the unstableLTA₄, the rate-limiting step in the synthesis of the leukotrienes, isalso catalyzed by 5-lipoxygenase. LTA₄ is then converted to either LTB₄or LTC₄. LTC₄ is rapidly metabolized to LTD₄ and then to LTE₄. LTC₄,LTD₄ and LTE₄ are collectively referred to as the cysteinyl (Cys)leukotrienes.

Biosynthesis of LTB₄, C₄, D₄ and E₄ occurs predominantly in leukocytes,in response to a variety of immunological stimuli. The primary target ofLTB₄ is the leukocyte where it elicits enzyme release, chemotaxis,adherence, and aggregation in nM concentrations. LTB₄ modulates immuneresponses and participates in the host-defense against infections.Hence, LTB₄ is an important chemical mediator in the development andmaintenance of inflammatory reactions and disease states.

In vitro evaluation of the ability of a composition to inhibit theenzymes 5- or 12/15 as described in Walidge, N. B. et al Anal. Biochem.,231: 354-358 (1995) using a high throughput assay format withcalorimetric method for the determination of lipoxygenase activity; aswell as in vitro evaluation of inhibiting LTB₄ is described in Examples.

Administration

The compounds of Formula I are administered at a therapeuticallyeffective dosage, e.g., a dosage sufficient to provide treatment for thedisease states previously described. Administration of the compounds ofthe invention or the pharmaceutically acceptable salts thereof can bevia any of the accepted modes of administration for agents that servesimilar utilities.

While human dosage levels have yet to be optimized for the compounds ofthe invention, generally, a daily dose is from about 0.01 to 10.0 mg/kgof body weight, preferably about 0.1 to 5 mg/kg of body weight, and mostpreferably about 0.3 to 1.0 mg/kg of body weight. The amount of activecompound administered will, of course, be dependent on the subject anddisease state being treated, the severity of the affliction, the mannerand schedule of administration and the judgment of the prescribingphysician.

In employing the compounds of this invention for treatment of the aboveconditions, any pharmaceutically acceptable mode of administration canbe used. The compounds of this invention can be administered eitheralone or in combination with other pharmaceutically acceptableexcipients, including solid, semi-solid, liquid or aerosol dosage forms,such as, for example, tablets, capsules, powders, liquids, suspensions,suppositories, aerosols or the like. The compounds of this invention canalso be administered in sustained or controlled release dosage forms,including depot injections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for the prolongedadministration of the compound at a predetermined rate, preferably inunit dosage forms suitable for single administration of precise dosages.The compositions will typically include a conventional pharmaceuticalcarrier or excipient and a compound of this invention or apharmaceutically acceptable salt thereof. In addition, thesecompositions may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, and the like, including, but not limited toanticoagulants, blood clot dissolvers, permeability enhancers and slowrelease formulations.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,preferably about 0.5% to 50%, by weight of a compound or salt of FormulaI, the remainder being suitable pharmaceutical excipients, carriers,etc.

One preferred manner of administration for the conditions detailed aboveis oral, using a convenient daily dosage regimen which can be adjustedaccording to the degree of affliction. For such oral administration, apharmaceutically acceptable, non-toxic composition is formed by theincorporation of any of the normally employed excipients, such as, forexample, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and the like. Such compositions take theform of solutions, suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations and the like.

Preferably the compositions will take the form of a pill or tablet andthus the composition will contain, along with the active ingredient, adiluent such as lactose, sucrose, dicalcium phosphate, or the like; alubricant such as magnesium stearate or the like; and a binder such asstarch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose andderivatives thereof, and the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier, such as, forexample, water, saline, aqueous dextrose, glycerol, glycols, ethanol,and the like, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, or solubilizing agents, pH buffering agents and thelike, for example, sodium acetate, sodium citrate, cyclodextrinederivatives, sorbitan monolaurate, triethanolamine acetate,triethanolamine oleate, etc. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 15th Edition, 1975. The composition or formulationto be administered will, in any event, contain a quantity of the activecompound in an amount effective to alleviate the symptoms of the subjectbeing treated.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 95% with the balance made up from non-toxic carrier may beprepared.

For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, talcum, cellulosederivatives, sodium crosscarmellose, glucose, sucrose, magnesiumcarbonate, sodium saccharin, talcum and the like. Such compositions takethe form of solutions, suspensions, tablets, capsules; powders,sustained release formulations and the like. Such compositions maycontain 0.01%-95% active ingredient, preferably 0.1-50%.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is preferablyencapsulated in a gelatin capsule. Such diester solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, thesolution, e.g. in a polyethylene glycol, may be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g. water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g. propylenecarbonate) and the like, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

Other useful formulations include those set forth in U.S. Pat. Nos. Re.28,819 and 4,358,603.

The formulation can be administered in a single unit dosage form forcontinuous treatment or in a single unit dosage form ad libitum whenrelief of symptoms is specifically required. For example, theformulation may be administered as a bolus or as a continuousintravenous infusion after onset of symptoms of stroke, myocardialinfarction or chronic heart failure.

Another preferred manner of administration is the topicaladministration. “Topical administration” refers to application of thepresent compositions by spreading, spraying, etc. onto the surface ofthe skin. The typical amount applied may vary from about 0.1 mg ofcomposition per square centimeter of skin to about 25 mg of compositionper square centimeter of skin. The compounds of the present inventionmay be formulated for topical administration to the epidermis asointments, creams or lotions or as transdermal patch. Formulationssuitable for topical administration in the mouth include lozenges,pastilles and mouthwashes.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, if desired, the pharmaceutical compositions to be administeredmay also contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents, solubilityenhancers, and the like, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate, cyclodextrins, etc.

A more recently devised approach for parenteral administration employsthe implantation of a slow-release or sustained-release system, suchthat a constant level of dosage is maintained. See, e.g., U.S. Pat. No.3,710,795. The percentage of active compound contained in suchparenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject. However, percentages of active ingredient of 0.01% to 10% insolution are employable, and will be higher if the composition is asolid which will be subsequently diluted to the above percentages.Preferably the composition will comprise 0.2-2% of the active agent insolution.

Nasal solutions of the active compound alone or in combination withother pharmaceutically acceptable excipients can also be administered.

Formulations of the active compound or a salt may also be administeredto the respiratory tract as an aerosol or solution for a nebulizer, oras a microfine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation have diameters of less than 50 microns, preferably less than10 microns.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

General Characterization Methods

As reported in the following examples, Nuclear Magnetic Resonance (NMR)spectra were recorded on a Bruker DTX 300 spectrometer using, in mostcases, tetramethyl silane (TMS) as the internal reference. Mass spectrawere obtained on an Agilent 1100 LC/MSD instrument using eitherelectrospray ionization (positive or negative mode) (ESI) or atmosphericpressure chemical ionization (positive or negative mode) (APCI).

Example 1 Beta-Amyloid Cell Death Assay Media Composition

Neurobasal/B27i: Neurobasal medium plus 1×B27 supplement, 0.5 mML-glutamine, 25 μM L-glutamic acid, and 0.5× Penicillin/StreptomycinNeurobasal/B27m: Neurobasal medium plus 1×B27 supplement and 0.5 mML-glutamineBSS (Ca/Mg free): HBSS (calcium/magnesium free) plus 10 mM Hepes (pH7.25), 1× Penicillin/Streptomycin, and 1 mM Sodium PyruvateGlucose-free BSS₀: 143.6 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mMMgSO₄, 1 mM NaH₂PO₄, 26.2 mM NaHCO₃, 10 mg/l phenol red, 0.25×Penicillin/Streptomycin, and 10 mM Hepes (pH 7.4)Papain Quench solution: Neurobasal medium plus 1×B27 supplement, 1×Penicillin/Streptomycin and 0.5 mg/ml DNase1Assay media: Neurobasal medium plus 1×B27 (minus AO) supplement, 0.5 mML-glutamine, and 0.25× Penicillin/Streptomycin.

Experimental Procedure Hippocampal Cell Culture

Hippocampal neurons were isolated from E18 rat embryos as follows.Embryos were decapitated and the heads immersed in cold BSS (Ca/Mgfree). Using a dissecting microscope the hippocampi were dissected outand placed in cold BSS (Ca/Mg free). The isolated hippocampi were thencentrifuged at 1000 rpm for 2 min, the BSS aspirated off and 2 ml of 2mg/ml Papain in Neurobasal media added per 10 embryos. After mixing on arotational shaker for 10 min at 37° C., 5 ml Papain Quench solution wasadded. Cells were then centrifuged at 1000 rpm for 2 min, thesupernatant was aspirated and 2 ml of Neurobasal/B27i was added. Thecells were triturated 6 times with siliconized pipettes (decreasing boresize) after which an additional 5 ml Neurobasal/B27i was added. The cellsuspension was then centrifuged at 1000 rpm for 2 min, the supernatantwas aspirated and 2 ml of Neurobasal/B27i was added. Cells weretriturated again as described above and the volume of Neurobasal/B27iwas adjusted to 1 ml/embryo. Cells were then counted and seeded at adensity of 75,000 cells per well in a poly-D-lysine coated 24-wellplate. After four days media was removed from the cells and replacedwith Neurobasal/B27m media+5 μM Ara-C (cytosine arabinoside). Seven daysafter isolation the media was removed again and replaced with freshNeurobasal/B27m media. Ten days after isolation the hippocampal cultureswere used in the assay described below.

Preparation of Oligomeric Beta-Amyloid (Aβ) Peptide

Aggregation of Aβ(1-42) (American Peptide Co, Sunnyvale, Calif.) intooligomers was carried out according to the method of Dahigren et al,(2002) Oligomeric and fibrillar species of amyloid-beta peptidesdifferentially affect neuronal viability. J Biol Chem 277: 32046-32053.The Aβ peptide was dissolved to 1 mM in hexafluoroisopropanol (HFIP) andaliquoted into sterile microcentrifuge tubes. The HFIP was removed undervacuum and the peptide film stored at −20° C. The day before the assay,the peptide film was resuspended in dry DMSO to a concentration of 5 mM.Ham's F-12 media was then added to bring the peptide to a finalconcentration of 100 μM, and this solution was incubated at 4° C. for 24hours to allow formation of oligomers.

Treatment of Hippocampal Neurons with Oligomeric Aβ

The existing growth medium was aspirated from the hippocampal culturesand the monolayer was washed once with 500 μl glucose free-BSS₀. Testarticles were diluted to 2-fold the desired testing concentration inassay media and 250 μL was added to the cells. From the 100 μMoligomeric beta-amyloid peptide solution described above, a workingsolution of 6 μM was made in assay media. 250 μL of this workingsolution was also added to the cells. The final volume for each well was500 μL and the final concentration of Aβ peptide was 3 μM. As a negativecontrol, cells were incubated with 500 μL assay media with no additions.

Cells were incubated in a 39° C. incubator (5% CO₂) for 24 hours. Afterthis time, the number of live neurons remaining in each well wasdetermined using a fluorescent vital cell stain, Cell Tracker Green(Molecular Probes, Eugene, Oreg.). Assay media was aspirated from thecells and 400 μL of 2.5 μM Cell Tracker Green was added to each well.Cells were placed in a 37° C. incubator for 5 minutes after which timethe cell stain was aspirated off and 500 μl of HBSS (Invitrogen, LifeTechnologies, Carlsbad, Calif.) was added to each well. The number oflive cells in each well was then quantitated using an automatedfluorescent microscope/imaging system (Universal Imaging, DowningtownPa.).

Certain compounds of the present invention such as

-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;    and-   2-[3-(Benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;    when tested as described above exhibited assay between 10% and 40%    protection in the β amyloid cell assay

Example 2 MPP⁺ Cell Death Assay Media Composition

RF media: DMEM-No glucose, glucose (29.1 mM), L-glutamine (1.4 mM), 10%heat-inactivated FBS, and 1× penicillin/streptomycin (P/S)Wash media: DMEM-No glucose and 1×P/SLow serum media: DMEM-No glucose, glucose (29.1 mM), L-glutamine (1.4mM), 0.5% FBS, and 1×P/SAssay Media: DMEM-No glucose, L-glutamine (1.4 mM), 0.5% FBS, and 1×P/S

Experimental Procedure

The substantia nigra-derived dopaminergic progenitor cell line wasseeded in poly-D-lysine-coated 24-well plates at a density of 4500 cellsper well in RF media. The cells were left to attach for 16 hours in a33° C. incubator (5% CO₂) after which time they were washed once with500 μL wash media and then differentiated into a neuronal phenotype byincubating in low serum media for 24 hours in a 39° C. incubator (5%CO₂).

After 24 hours the low serum medium was aspirated from the cells and themonolayer was washed once with 500 μL wash media. Test articles werediluted to 2-fold the desired testing concentration in assay media and250 μL was added to the cells. From a 10 mM stock, a working solution of140 μM 1-methyl-4-phenylpyridinium (MPP⁺) (Sigma, St. Louis, Mo.) wasmade in assay media and 250 μL of this working solution was also addedto the cells. The final volume in each well was 500 μL and the finalconcentration of MPP⁺ was 70 μM. As a negative control, cells wereincubated with 500 μL assay media with no additions.

Cells were incubated in a 39° C. incubator (5% CO₂) for 24 hours. Afterthis time, the number of live neurons remaining in each well wasdetermined using a fluorescent vital cell stain, Cell Tracker Green(Molecular Probes, Eugene, Oreg.). Assay media was aspirated from thecells and 400 μL of 2.5 μM Cell Tracker Green was added to each well.Cells were placed in a 37° C. incubator for 5 minutes after which timethe cell stain was aspirated off and 500 μL of HBSS (Invitrogen LifeTechnologies, Carlsbad, Calif.) was added to each well. The number oflive cells in each well was then quantitated using an automatedfluorescent microscope/imaging system (Universal Imaging, DowningtownPa.).

Results:

Certain compounds of the present invention such as

-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   [3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonic    acid dimethyl ester;-   3-[6-Hydroxy-2-methyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl]-propionic    acid;-   2-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-1-morpholin-4-yl-propan-1-one;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-[3-(Benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(2-Chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   2,6:9,12-Dimethano-9,10,11,12-tetrahydro-2,4,4-trimethylnaphtho[1,2-b]oxocan-8-ol;-   5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-2,2,7,8-tetramethyl-chroman-6-ol;-   2,2-Dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   5-Bromo-2,2-dim    ethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-7,8,9,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;    and-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol    when tested as described above provided protection in at least 30%,    preferably in at least 50% of the cells tested at concentrations    ranging from 1 to 25 μM.

Example 3 MPTP Animal Model

Male C57/BL6 mice (Harlan, Ind.), weight 25-30 g, were used in allstudies. MPTP-HCl (Sigma) was administered i.p. according to one of thefollowing protocols. The maximum volume which was given per injection is200 μL. In all studies, animals were euthanized with carbon dioxide and,brains removed for subsequent determination of dopamine depletion whereappropriate.

Subacute Model

Animals received 25 mg/kg of MPTP once a day for 5 consecutive days. Theend point was 2 days after the final dose.

Acute Model

Animals received 4×20 mg/kg of MPTP at 2 hour intervals. The end pointwas at either 7 or 14 days.

Subchronic Model

Animals received 2×40 mg/kg of MPTP with this repeated 16 hours later.The end point was at either 14 or 28 days.

Chronic Model

Animals received 25 mg/kg of MPTP, given twice weekly for 5 weeks. Theend point was either 1, 3 or 24 weeks after the final dose.

Neurobehavioral Outcome Measures

Compound efficacy was examined with the use of neurobehavioral models.These models allowed the determination of a given compound's ability toreverse the motor deficits seen with MPTP treatment.

All animals received pre-training for the individual model and abaseline reading was obtained one day prior to the commencement of MPTPtreatment.

Open Field Test

The open field test measures spontaneous activity. Deficits can beobserved with hippocampal and basal ganglia lesions, and with hind limbdysfunction. It is sensitive to moderate dopamine (DA) depletion.

The open field test was carried out in a clear, acrylic open box 60cm×60 cm. The base of the box was marked into a 5×5 grid of 12 cm×12 cmsquares. Animals were individually placed in the box and allowed to roamfree. The number of squares an animal crossed in a 90 second time periodwas recorded. In order to be scored, the animal must either have had allfour limbs within a given square or, all four limbs must have left agiven square.

Tactile Adhesion Model

The tactile adhesion model measures an animal's ability to complete acomplex sensorimotor task. It is sensitive to moderate to severe DAdepletion.

A tactile stimulus (0.5×0.5 cm square of “sticky tape”) was applied toeach side of the animal's face and the time taken to remove it wasrecorded. The following measurements were recorded:

a. Latency to contact left side tape

b. Latency to contact right side tape

c. Order of side contacted (left vs. right)

d. Latency to remove left side tape

e. Latency to remove right side tape

Pole Test

The pole test evaluates motor co-ordination.

A rough surfaced metal pole (diameter 8 mm, height 50 cm) protrudingfrom a cage filled with animal bedding was used for this test. Animalswere placed head upwards at the top of the pole. They were required toturn and descend face downwards and the time taken for this was recorded(latency to reach bedding). Timing was started when the individualanimal gripped the top of the pole and was stopped when all four limbscontacted the bedding.

Compound Administration

All test compounds were administered ip. The maximum volume which wasgiven per injection was 200 μL. Compounds were dosed up to twice daily,two days prior and 7 days post MPTP treatment.

Data Analysis

A baseline reading was taken for each animal one day prior to MPTPtreatment. All subsequent readings were normalized to the individualanimal's baseline. Values were expressed as a percent baseline.

Data is percent baseline and expressed as mean±std dev:

Day 1 Day 2 Day 3 MPTP positive 1329 ± 973 2740 ± 1298 1641.7 ± 651.1 control Neg control  90.8 ± 27.4 98.9 ± 42.5 137.2 ± 100.9 (no MPTP)L-DOPA   110 ± 13.2 97.9 ± 20.0 100.1 ± 14.8  Vehicle 1238.3 ± 987.81008.5 ± 992.1  308.9 ± 239.6  3 mg/kg/day  623.7 ± 533.1  355.3 ±207.3*  90.7 ± 15.2* 10 mg/kg/day 520.2 ± 52.1 363.7 ± 94.9* 137.9 ±44.9* 30 mg/kg/day  105.3 ± 22.1*†  94.2 ± 27.7*† 108.2 ± 29.1* *p <0.05 (relative to MPTP induced deficit) †p < 0.05 (relative to vehicle)

Summary

Compound and vehicle given for 9 days in total, 2 days prior to MPTP and7 days after MPTP treatment. L-DOPA given 1 hour prior to the pole testeach day.

Results

Certain compounds of Formula I, such as

-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid    at a dose of 30 mg/kg/day significantly reduced the functional    deficit produced by MPTP on each of days 1-3. Functional outcome was    significantly better on days 1 and 2 than in vehicle treated animals    and in both cases was comparable to that of the negative control    group.

Doses of 10 mg/kg/day and 3 mg/kg/day significantly reduced the MPTPinduced deficit on days 2 and 3 but this was not significantly differentfrom vehicle treated animals.

Example 4 FRDA Fibroblast Assay for Protection from Oxidative Stress A.Cell Culture and Reagents

Primary fibroblasts were derived from donors with a molecular diagnosisof FRDA and control donors with no mitochondrial disease. Lines F2, C2and C3 were obtained from Coriell Cell Repositories (Camden, N.J., USA;catalog #'s GM04078, GM 08402 and GM08399, respectively). All cell typeswere diagnosed at the molecular level for intronic GAA triplet repeatlength in the frataxin gene using a PCR-based method, according tomethods known in the art. FRDA-fibroblasts types had ˜400-450 repeats(F2 line) or more (F1 and F3), whereas control cell lines displayedrepeats of normal length. The cells were seeded in 96-well plates at adensity of 4000 cells per 100 μl in growth medium consisting of 25%(v/v) M199 EBS and 64% (v/v) MEM EBS without phenol red (Bioconcept,Allschwil, Switzerland) supplemented with 10% (v/v) fetal calf serum(PAA Laboratories, Linz, Austria), 100 U/ml penicillin, 100 μg/mlstreptomycin (PAA Laboratories, Linz, Austria), 10 μg/ml insulin (Sigma,Buchs, Switzerland), 10 ng/ml EGF (Sigma, Buchs, Switzerland), 10 ng/mlbFGF (PreproTech, Rocky Hill, N.J., USA) and 2 mM glutamine (Sigma,Buchs, Switzerland). The cells were incubated in the presence of thevarious test compounds for 24 h before addition of 1 mM BSO(L-buthionine (S,R)-sulfoximine).

B. Cell Viability Measurements

Cell viability was measured after the first signs of toxicity appearedin the BSO-treated controls (typically after 16-48 h). The cells werestained for 60 min at room temperature in PBS with 1.2 μm calceinAM and4 μm ethidium homodimer (Live/Dead assay, Molecular Probes, Eugene,Oreg., USA). Fluorescence intensity was measured with a GeminiSpectramax XS spectrofluorimeter (Molecular Devices, Sunnyvale, Calif.,USA) using excitation and emission wavelengths of 485 and 525 nm,respectively.

C. Data and Statistics

In experiments carried out in support of the present invention, certaincompounds such as

-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-[6-Hydroxy-2-methyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl]-propionic    acid;-   2-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,6:9,12-Dimethano-9,10,11,12-tetrahydro-2-methylnaphtho[1,2-b]oxocan-8-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(2-Chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   5-Bromo-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   2-(3-Isobutylsulfanyl-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiophen-2-yl-3,4,7,8,9,10-hexa    hydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-7,8,9,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol    significantly reduced cell death in FRDA fibroblasts compared to    untreated FRDA fibroblasts with an EC₅₀ of between 0.01 μM and 10    μM.

Example 5 Determination of Activity Utilizing Neuronal Cell Stress AssayA. Isolation and Culture of Primary Hippocampal Neuronal Cells.Materials

-   -   Neurobasal/B27: Neurobasal medium (Invitrogen, Carlsbad, Calif.)        with 1×B27 supplement (Invitrogen Life Technologies), 0.5 μM        L-glutamine, 25 μM L-glutamic acid, and 1×        Penicillin/Streptomycin.    -   Hank's Basic Salt Solution (HBSS, Ca/Mg-free) was prepared by        preparing 1× Hanks CMF (Gibco) supplemented with HEPES (10 mM,        pH 7.3), sodium bicarbonate (0.35%), 1× Penicillin/Streptomycin,        and 1 mM pyruvate.    -   Poly-D-lysine (Sigma, St. Louis, Mo.), 50 μg/ml solution        filtered through 0.2 μm filter tubes.    -   Sigmacote (Sigma, St. Louis, Mo.).    -   Plastic Culture Flasks (T75 cm²) or 12-well cell culture plates        treated with Poly-D-Lysine (Sigma, St. Louis, Mo.).

Preparation of Primary Hippocampal Neuronal Cells

A pregnant female mouse (E18-E19) was euthanized with CO₂ prior toremoval of the uterus, which was then placed in a sterile plastic petridish. The embryos were removed from the sac, and the embryonic brainswere removed and immersed in cold (4° C.) Buffered Salt Solution (HBSS;Ca/Mg free; Invitrogen Life Technologies) in a small petri dish.Hippocampi were then removed from the brains under a dissectingmicroscope and were placed on a paraffin-covered dish. The meninges werestripped away and the dissected hippocampi were collected in a smallpetri dish in HBSS. The hippocampi were transferred to a 15-mlcentrifuge tube (normally 10-12 brains) filled with HBSS. The tubecontaining the brains was centrifuged at 1000 rpm for 2 min in atabletop centrifuge. The supernatant was removed, 2 ml of HBSS was addedto the hippocampi in the tube, and the resulting suspension wastriturated 2 times each with long-tipped siliconized glass pipetteshaving progressively smaller apertures, starting with a pipette with astandard size opening (approximately 1.0 mm diameter), following withone having an aperture of half standard size (approximately 0.5 mmdiameter), then with one having an aperture about one-half that size(0.25 mm diameter). The suspension was then centrifuged again at 1000rpm for 2 min in a tabletop centrifuge, the supernatant was discarded,and 2 ml of Neurobasal/B27i (with antibiotics) was added to the tube.The trituration procedure described above was then repeated on thissuspension.

The density of cells was determined on a small aliquot of cells usingstandard counting procedures and correcting for cell viability by trypanblue stain exclusion. Using this procedure, the expected yield is3×10⁵-6×10⁵ cells/brain. Cells were then added to PDL-coated 24-wellplates, flasks or MetTek dishes in Neurobasal/B27I at a density of about1.5×10⁶ cells (T75 flask) or about 70,000 cells/well of a 24-well plate.Plated cells were incubated at 37 degrees in an atmosphere of 5% CO₂/95%O₂. Media was renewed after 3-4 days by replacing half of it with freshNeurobasal/B27m medium, containing 5 μM cytosine arabinoside (AraC).Seven to eight days from the initial culture, the media was renewedagain, by removing one-half or it and replacing with an equal amount offresh Neurobasal/B27m medium (without Ara-C).

B. Hippocampal Anoxia-Reoxygenation Cell Death Assay.

This assay was used to induce ischemia by anoxia-reoxygenation incultured hippocampal neuronal cells. Test compounds were added to assesspotency and efficacy against ischemia-induced neuronal cell injury andcell death.

Materials.

-   -   Neurobasal media, NoG neurobasal media, B27 supplement and B27        Supplement minus AO were obtained from Invitrogen Life        Technologies.    -   Neurobasal/B27 medium was prepared with 2×B27 minus AO        supplement, 0.5 mM L-glutamine and 0.25×        penicillin/streptomycin.    -   Cell Tracker Green was obtained from Molecular Probes and a        fresh 5 μM solution was prepared from 10 mM stock just before        use.    -   LoG-Neurobasal contains NoG neurobasal medium plus 1 mM glucose,        0.5 mM L-glutamine, 0.25× Penicillin/Streptomycin, and 10 mM        Hepes (pH 7.4).    -   Primary hippocampal neuronal cells were prepared according to        the methods described above and were cultured in poly-D-lysine        coated 24-well plates for 10-11 days prior to use.

Deoxygenated LoG-Neurobasal medium (100 ml) was prepared bypre-equilibrating the medium in a T150 cm² flask in a hypoxic chamberovernight. Following pre-incubation under hypoxic conditions, theLoG-Neurobasal media was lightly bubbled with 100% N₂ for 30 min tocompletely deoxygenate the media. An additional 20 ml LoG-Neurobasal waspre-equilibrated in a T75 cm² flask and was incubated in a normalincubator (5% CO₂) overnight. Reoxygenated medium was prepared byplacing Neurobasa/B27 media overnight in the culture incubator (5%CO₂/95% O₂).

10-11 Days after plating the hippocampal neurons, existing culturemedium (Neurobasal/B27m) was removed from the cells by aspiration. Cellswere washed once with 600 μl/well (24-well culture plates) of glucosefree-BSS. Neurons were replenished with deoxygenated LoG-Neurobasal (400μl per well for each well of a 24-well plate). Test compounds were addeddirectly to each well (usually 3 concentrations of the compound pluspositive control, each in triplicate). Most test compounds weredissolved in 100% DMSO; however, concentrations were adjusted such thatthe final concentration of DMSO in the cell media never exceeded 0.5%.Plates containing cells with test compounds were placed in a hypoxicchamber for 4-5 hr with plate lids ajar. For normoxia controls,pre-equilibrated normoxic LoG-Neurobasal medium was added to each wellof cells, and the plate was replaced in the normal culture incubator for4-5 hr. After 4-5 hr of hypoxia, the existing media was carefullyaspirated off, and 400 μL of new, reoxygenated (pre-equilibrated)Neurobasal/B27 was added to each well. The same test compounds (in thesame the concentrations) were added back into the corresponding wells.Plates were placed in the cell culture incubator (5% CO₂/95% O₂) andreoxygenated for 20-24 hr. After reoxygenation for 20-24 hr, liveneurons were quantitated using the cell tracker green fluorescencemethod, described below.

To test for cell viability, existing culture medium was aspirated fromeach well of the 24 well plates, and neurons were washed once with 1 mLof HBSS (pH 7.4, pre-warmed to 30-37° C.). To each well was added 500 μLof 5 μM Cell Tracker Green fluorescent dye dissolved in HBSS. Plateswere placed in the dark at room temperature for 15 minutes, then werewashed with 1 mL of HBSS. 500 μL of HBSS was then added to each well,and fluorescent cells were counted using a fluorescent microscope.Significantly increased cell viability compared to control cells isindicative of a protective compound.

Certain compounds of the present invention such as:

-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   [3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonic    acid dimethyl ester;-   [3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   4-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-butane-1-sulfonic    acid dimethylamide;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   3-[7-(2-Methoxycarbonyl-ethyl)-2,7-dimethyl-2,7,9,10,11,12-hexahydro-1,8-dioxa-0-12-methano-triphenylen-2-yl]-propionic    acid methyl ester-   3-[6-Hydroxy-2-methyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl]-propionic    acid;-   2-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-6-ol;    and-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;    when tested as described above provided protection against    stressor-induced cell death in at least about 30% of the cells    tested, at concentrations ranging from 1 to 100 μM, preferably at    concentrations of 25 μM.

Example 6 Rat Middle Cerebral Artery Occlusion (MCAO) Model of CerebralIschemia A. Animal Preparation

Male Wistar rats (Harlan, Ind.) weighing 300-350 g were commonly used inthese experiments. Animals were allowed free access to water andcommercial rodent diet under standard laboratory conditions. Roomtemperature was maintained at 20-23° C. and room illumination is on a12/12-hour light/dark cycle. Animals were acclimatized to the laboratoryenvironment 5 to 7 days prior to the study, and fasted (with free accessto water) overnight before surgery.

B. Middle Cerebral Artery Occlusion (MCAO)

Anesthesia was maintained by inhalation of 3.0% isoflurane (Aerrane,Front Dodge, Iowa) in 0.8% oxygen. The animal's neck was shaved andsterilized before operation. Body temperatures were controlled andmaintained at 37.5° C.+/−1 degree via external heating and coolingdevices. To lower the body temperature, animals were placed in a coolingchamber, which uses ice to cool circulating air. Throughout the studythe body temperature was recorded using a temperature transponder (BMDSInc., Seaford, DL) implanted subcutaneously at the time of MCAO betweenthe rat shoulder blades that allowed the user to read the bodytemperature via a pocket scanner (BMDS Inc., Seaford, DL). The bodytemperature was taken by inserting the temperature probe into theanimal's rectum. Body temperature was recorded every hour for 6 hourspost-occlusion; however, body temperatures were taken more frequently sothat they could be maintained at the normothermic temperature.

Animals were subjected to two hours MCAO using a modified intraluminalfilament technique, as follows: A midline incision on the ventral partof the neck was made to expose external and internal carotid arteries.The right external and common carotid arteries were ligated by a suture(silk 5/0, Carlisle Laboratories, Farmers Branch, Tex.) and the rightinternal artery was temporarily ligated using a microvascular clip (FineScience Tool Inc., Foster City, Calif.). A small incision was made inthe common carotid artery. A nylon filament, its tip rounded by heating,was prepared from a fishing line (Stren Fishing Lines, Wilmington, Del.)and was inserted from the right common carotid artery. The filament wasadvanced into the internal carotid artery 18-20 mm from the point ofbifurcation of internal and external arteries and a suture is tightlyligated around the filament. Two hours post occlusion, animals werere-anesthetized to allow reperfusion for the remaining of the experimentby removal of the filament.

C. Drug Administration

Test compounds may be administered by any of a number of routes, such asthose described below. Compounds can be administered before, during orafter occlusion, as appropriate to the protocol.

a) Intracerebroventricular (ICV) Infusion

The anesthetized animal is placed on a stereotaxic apparatus (HarvardApparatus, S. Natick, Mass.). Anesthesia is maintained by inhalation of3.0% isoflurane (Aerrane, Front Dodge, Iowa) in 0.8% oxygen throughoutthe entire procedure. The scalp is shaved and sterilized prior tosurgery. A midline sagittal incision about 3 cm long is made slightlybehind the eyes to expose the skull. The skull is scraped with a roundedend spatula to remove periosteal connective tissue. A bur hole is placed1.5 mm lateral, 1 mm posterior to the left of the bregma to mark theleft lateral ventricle. A brain infusion cannula (ALZET Co., Palo Alto,Calif.) is inserted 4 mm deep into the hole. The desired depth isadjusted by attaching spacers to the cannula. The cannula attached to a4-cm silastic catheter (Helix Medical Inc., Carpinteria, Calif.) fixedin place with dental cement (Ketac-cement, Norristown, Pa.). Thecatheter is either attached to a primed osmotic pump placedsubcutaneously between the shoulder blades for permanent infusion or toa syringe for a short infusion.

b) Intravenous (IV) Osmotic Pump Implantation into the Jugular Vein

Anesthesia is maintained by inhalation of 3.0% isoflurane (Aerrane,Front Dodge, Iowa) in 0.8% oxygen throughout the entire procedure. Theanimal's neck will be shaved and sterilized before operation. A midlineincision is made on the ventral part of the neck to exposes the jugularvein. The vein is isolated and ligated with a suture (silk 5/0, CarlisleLaboratories, Farmers Branch, Tex.) rostral to the point of the incisionand a microvascular clip (Fine Science Tool Inc., Foster City, Calif.)close to the heart. A small incision is made between two ligations. A2-cm silastic catheter (Helix Medical Inc.) attached to a PE-60 tube(Becton. Dickinson and Co. Sparks, Md.) connected to an ALZET (ALZET Co.Palo Alto, Calif.) pump is introduced and advanced 2 mm into the jugularvein toward the heart. The microvascular clip is removed and thecatheter is secured in place with a suture (silk 5/0, CarlisleLaboratories, Farmers Branch, Tex.). The pump is placed into a pocketmade subcutaneously between the shoulder blades, allowing the catheterto reach over neck to the jugular vein with sufficient slack to permitfree movement of neck and head.

c) IV Infusion Via Femoral Vein

Anesthesia is maintained by inhalation of 3.0% isoflurane (Aerrane,Front Dodge, Iowa) in 0.8% oxygen throughout the entire procedure. Theexterior site of the right femoral vein is shaved and sterilized priorto surgery. A 3-cm incision is made in the right groin region and thefemoral vein is isolated. A small incision is made on the femoral veintemporarily ligated with a microvascular clip to introduce and advance apolyethylene (PE-50) catheter (Becton Dickinson and Co. Sparks, Md.).The catheter is secured in place with suture (silk 5/0, CarlisleLaboratories, Farmers Branch, Tex.). The other end of the catheter isattached to a syringe filled with the heparinized saline for the bolusinjection. Using a hemostat, a pocket is made subcutaneously on the backof the animal so the PE catheter can be brought up to theexteriorization point at the nape of the neck for either a bolusinjection or a continuous injection by an osmotic pump.

d) Intraperitoneal (IP) Injection

An awake rat is held in a standard hand hold position, a 23¾G needle isinjected into the lower right quarter of the abdomen pass theperitoneum, slightly off the midline. To avoid organ injection, theplunger of the syringe is slightly pulled back. If no fluid iswithdrawn, the content of the syringe is delivered into the abdominalcavity.

e) Gavage Feeding

A standard rat gavage tube (Popper & Sons Inc., NY) is attached to a3-cc hypodermic syringe. The animal is held by the shoulder in avertical position. The feeding tube is placed into the mouth thenadvanced until it reaches the stomach (the approximate insertion lengthof the tube was measured prior to the feeding). The content of thesyringe is slowly delivered, and then the tube is withdrawn.

D. Behavioral Assessment

One hour after MCAO, the animal was gently held by its tail and observedfor forelimb flexion. Then the animal is placed on the floor to beobserved for walking pattern; only the animals that score 3 on Bedersongrading system (Table 1) are included in the study.

TABLE 1 Bederson Grading System for Neurological Evaluation Neurologicaldeficit Grading Behavioral observation Normal grade 0: No observabledeficit Moderate grade 1: forelimb flexion Severe grade 2: forelimbflexion, decreased resistance to lateral push Extreme grade 3: forelimbflexion, decreased resistance to lateral push, circle to paretic side

E. Evaluation of Ischemic Damage

Twenty-four hours post-MCAO, or longer, in some experiments, animalswere sacrificed by CO₂ asphyxiation (dry ice). The brain was quicklyremoved from the skull, using standard procedures, rinsed in chilledsaline solution, and placed on a rat brain tissue slicer (ASIinstrument, MI). Seven 2-mm thick coronal slices are cut from each brainusing razor blades. The slices were immersed in 0.9% saline containing1.0% 2,3,5-triphenyltetrazolume chloride (TTC) (Sigma Chemical Co., St.Louis, Mo.) and incubated in a 37° C. water bath for 30 minutes.

After staining, each 2-mm slice was photographed with a TMC-7 camera (JHTechnologies, Ca) which were directly connected to a desktop PC tocapture and saved the image of each brain slice. This image was used forthe measurements of the regions of interest using a computer-based imageprocessing system (Metamorph).

To measure each area, the region of interest was selected using afreehand selection tool, the area was automatically computed byselecting the measure command. The measurements for primary regions ofinterest were right hemisphere, left hemisphere, total infarct,subcortical infarct, total penumbra and subcortical penumbra. After allregions of interest were measured for all seven slices of the brain,they were sorted by slice number and the corresponding regions ofinterest using a custom made Excel™ macro. This macro calculates thecortical penumbra, cortical infarct and total ischemic damage for eachslice; the corresponding areas of each rat brain were added together toproduce a single measurement for each area. Since the ipsilateralhemisphere is swollen following MCAO, edema volume was calculated andreported as the volumetric differences between the right and lefthemispheres of each brain slice. Using the % of hemispheric swelling allthe volumes were corrected for the edema.

The volume of the damage was determined using the calculations below foreach rat's brain.

Measurement Equation Corrected Value(s) Cortical Penumbra Total Penumbra(T.P.) − T.P._(corr)) = (T.P. × % H.S./100) (C.P.) Subcortical Penumbra(S.P.) C.P._(corr.) = C.P. − (C.P. × % H.S./100) S.P._(corr.) = S.P. −(S.P. × % H.S./100) Cortical Infarct Total Infarct (T.I.) − T.I._(corr.)= T.I. − (T.I. × % H.S./100) Subcortical Infarct (S.I.) S.I._(corr.) =S.I. − (S.I. × % H.S./100) C.I._(corr.) = C.I. − (C.I. × % H.S./100)Total Ischemic Total Penumbra + T.I.D._(corr.) = T.I.D. − (T.I.D. × %H.S./100) Damage (T.I.D.) Total Infarct Total Volume Each value ismultiplied by 2 (the thickness of the tissue). (mm³) Edema Volume Thevolumetric differences between the sum of right and left hemispheresdetermines the edema volume. % Hemispheric Edema × 100/left swelling(H.S.) hemisphere

F. Statistical Analysis

Sample size was chosen to achieve a 90% probability of significantresults. The measurements, which represented the same region of interestin seven slices of each rat's brain were added together to yield asingle measurement for total infarct, subcortical infarct, corticalinfarct, total penumbra, subcortical penumbra, cortical penumbra, totalischemic damage and edema in each animal. Group data was presented asmeans+/−SEM. Differences at the level of p<0.05 were consideredstatistically significant. Between groups comparison of each region ofinterest are carried out by unpaired student t test (between two groups)or one way ANOVA followed by post hoc Bonferroni's multiple comparisonsor by the nonparametric Dunnett's test (between control and the drugtreated groups).

Test compounds of the present invention were administered by intravenousosmotic pump implantation, and IV infusion. Certain compounds of thepresent invention such as:

-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    when tested as described above provided a reduction in total infarct    volume of at least about 30% at doses in the range of 10 μg/kg to 40    mg/kg.

Example 7 Interleukin-1β Microglial Cell Assay Materials and EquipmentA. Materials for Cell Preparation and Experiment

-   -   Mouse microglial cell line    -   DMEM High Glucose media (Gibco Catalog # 11965-092)    -   FBS (Hyclone Catalog # SH30070.03)    -   100× Penicillin/Streptomycin (Gibco Catalog # 15140-122).    -   LPS (Sigma Catalog # L2537)    -   Interferon-gamma (Sigma Catalog # I4777)    -   Cell Tracker Green (Molecular Probes Catalog # C2925)    -   HBSS buffer (950 ml Pyrogen-free water, 2.44 g/L MgCl2.6H20,        3.73 g/L KCl, 59.58 g/L Hepes, 58.44 g/L NaCl, 1.36 g/L KH2PO4,        1.91 g/L CaCl2.2H2O and pH to 4.5 with HCl)    -   Sterile 96-well plates precoated with poly-D-lysine (Corning        Catalog # 3665)    -   96-well deep well mother plate, DyNA Block 1000 (VWR Catalog #        40002-008)        B. Materials for Il-1beta Elisa    -   Mouse IL-1 beta Duo Set (R & D Systems Catalog # DY401)    -   Substrate Solution (R & D Systems Catalog # DY 999)    -   Bovine Serum Albumin fraction V (BSA V) (Sigma Catalog # A4503)    -   96-well Costar EIA high binding plates (VWR Catalog # 29442-302)    -   Plate seal (VWR Catalog # 29442-310)    -   PBS (Irvine Scientific Catalog # 9240)    -   Cell Culture Grade Water (Irvine Scientific Catalog # 9312)    -   Tween 20 (Sigma Catalog # P 1379)    -   Sucrose (Sigma Catalog # S7903)    -   Sodium Azide (Sigma Catalog # S 8032)    -   H₂SO₄ 5N (VWR Catalog # JT 5691-2)

Experimental Preparation and Procedure:

LPS Activation:

Mouse microglial cells were seeded in poly-D-lysine coated 96-wellplates at a density of 10,000 cells/well and allowed to attach for 24hours. Cells were stimulated by addition of LPS (10 μg/ml) and IFN gamma(10 ng/ml) in the presence or absence of test article. The cells werethen incubated for 24 hours at 37° C., after which time the media wasremoved and used for cytokine determination as described below.

Cell Viability:

Viability of mouse microglial cells after exposure to the test articlewas determined using a fluorescent viability dye, Cell Tracker Green.Cell Tracker Green was used at a working concentration of 5 μM in1×HBSS. Cells were washed once with HBSS (200 μl/well) and 100 μl CellTracker Green was added to each well. Cells were then incubated at 37°C. for 30 minutes, after which time the Cell Tracker was removed and thecells were washed once with HBSS (200 μl/well). 100 μl fresh HBSS wasadded to each well and the plate was read on a Fluoroskan plate readerusing an excitation wavelength of 485 nm and an emission wavelength of538 nm.

Mouse IL-1beta Elisa:

Solutions:

Wash Buffer: PBS 1 L+500 μl Tween 20 (final 0.05%) pH 7.2-7.4.

Blocking Buffer: 500 ml PBS+5 g BSA V (1%)+25 g Sucrose (5%)+0.25 gSodium Azide (0.05%).

Reagent Diluent: 500 ml PBS+5 g BSA V (1%) pH 7.2-7.4 and filtersterilize through 0.2 μm.Stop Solution: 2N sulfuric acid.

Duo Set Preparations:

1. The IL-1β capture antibody was reconstituted in 1 ml of PBS to give afinal concentration of 720 μg/ml, and the working concentration was 4μg/ml. For coating one 96-well plate (at 100 μl/well) 56 μl of the 720μg/ml stock was diluted into 10 ml of PBS.2. The IL-1β standards were reconstituted in 0.5 ml of Reagent Diluent(70 ng/ml). For a high standard of 1 ng/ml (2 wells at 100 μleach+enough for series dilution) 7.1 μl of the 70 ng/ml standard werediluted into 0.5 ml of Reagent Diluent3. The IL-1β detection antibody was reconstituted in 1 ml of ReagentDiluent to give a final concentration of 18 μg/ml and the workingconcentration is 100 ng/ml. For one 96-well plate (at 100 μl/well) 56 μlof the 18 μg/ml stock was diluted into 10 ml of Reagent Diluent.

IL-1.beta ELISA Procedure:

Plate Preparation:

-   -   The Costar EIA Hi-binding plate was coated with capture antibody        at 4 μg/ml. Each well was coated with 100 μl, and the plate was        sealed and incubated overnight at room temperature.    -   Each well was aspirated and washed 3× with Wash Buffer. Each        well was filled to the top, dispensed, and any remaining buffer        was removed by inverting the plate and gently blotting against        clean paper towels.    -   Non-specific binding sites were blocked by adding 300 μl of        Blocking Buffer to each well, and after sealing incubating for        at least 1 hour at room temperature.    -   After washing the plate was now ready for the samples.

Assay Procedure:

-   -   100 μl of either standard or sample were added in each well of        the capture-coated and pre-blocked plate. The plate was sealed        and incubated for 2 hours at room temperature, followed with        washing.    -   100 μl of the detection antibody (10 ng/ml) were added to each        well. The plate was sealed and incubated at room temperature for        2 hours, followed with washing.    -   100 μl of the working dilution of Streptavidin-HRP was added,        and the plate was sealed and incubated in the dark for 20        minutes at room temperature, followed with washing.    -   The fresh Substrate Solution was prepared by mixing Color        Reagent A (H₂O₂) and Color Reagent B (Tetramethylbenzidine) in a        1:1 ratio. 100 μl of this Substrate Solution mixture was added        to each well and the plate was incubated in the dark for 20        minutes at room temperature.    -   50 μl of Stop Solution was added to each well, mixing was        ensured by gently tapping.    -   Each plate was read with the Spectramax once at 450 nm.

Results

When tested as described above, compounds of the present invention, suchas:

-   3-[7-(2-Methoxycarbonyl-ethyl)-2,7-dimethyl-2,7,9,10,11,12-hexahydro-1,8-dioxa-0-12-methano-triphenylen-2-yl]-propionic    acid methyl ester;-   3-[6-Hydroxy-2-methyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl]-propionic    acid;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic    acid;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester; and-   2,2-Dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;    inhibited IL-1beta production with an EC₅₀ of 20 μM or less.

Example 8 Rat Paw Edema Assay Animal Preparation:

Male Sprague-Dawley rats weighing between 175 to 200 g are used in thisstudy. Animals are allowed free access to water and commercial rodentdiet under standard laboratory conditions. Room temperature ismaintained at 20-23° C. and room illumination is on a 12/12-hourlight/dark cycle. Animals are acclimatized to the laboratory environment5 to 7 days prior to the study.

Experimental Procedure:

Each animal was treated by administration of vehicle, reference or testsubstance one hour prior to carrageenan injection, as follows:

I.V. Infusion via Femoral Vein: Anesthesia was maintained by inhalationof 3.0% isoflurane (Aerrane, Front Dodge, Iowa) in oxygen throughout theentire procedure. The exterior site of the right femoral vein was shavedand sterilized prior to surgery. A 3-cm incision is made in the rightgroin region and the femoral vein was isolated. The femoral vein wastemporarily ligated with a micro-vascular clip, and a small incision wasmade on the femoral vein to introduce and advance a polyethylene (PE-50)catheter (Becton. Dickinson and Co., Sparks, Md.). The catheter wassecured in place with suture (silk 5/0, Carlisle Laboratories, FarmersBranch, Tex.). The other end of the catheter was attached to a syringefilled with the saline for the bolus injection. Using a hemostat, apocket was made subcutaneously on the back of the animal so the PEcatheter could be brought up to the exteriorization point between theshoulder blade for either a bolus injection or a continuous injection byan osmotic pump.

I.P. Injection: An awake rat was held in a standard hand held position.A 23¾G needle was injected into the lower right quarter of the abdomenpass the peritoneum, slightly off the midline. To avoid organ injection,the plunger of the syringe was slightly pulled back. If no fluid iswithdrawn, the content of the syringe was delivered into the abdominalcavity.

Gavage Feeding: A standard rat gavage tube (Popper & Sons Inc, NY) wasattached to a 3-cc hypodermic syringe. The animal was held in a verticalposition. The feeding tube was placed into the mouth and then gentlyadvanced until it reached the stomach (the approximate insertion lengthof the tube should be measured prior to feeding). The content of thesyringe was slowly delivered, and then the tube was withdrawn.

One hour post treatment each animal was anesthetized with 3.0%isoflurane (Aerrane, Front Dodge, Iowa) in oxygen and administered 100μl of 1% Carrageenan Lambda type IV (Sigma Chemical Company, St. Louis,Mo.) suspension in saline, into the intraplantar surface of the righthind paw. Paw edema was measured four hours after carrageenan injection,either by measuring the increase in paw volume using a plethysmometer orthe increase in paw weight using a fine scale. Immediately prior toedema measurement, the animals were euthanized via CO₂ asphyxiation and500 μl blood was withdrawn by cardiac puncture for later analysis. Pawvolume is determined by the extent to which water was displaced by thepaw from a pre-calibrated chamber. The volume of the left hind paw(control) was subtracted from the volume of the right hind paw(carrageenan-treated) to determine the volume of carrageenan-inducededema. To measure the weight difference between paws, both hind pawswere removed and weighed separately.

To minimize the variation in the model following steps were taken:

-   -   Carrageenan was made fresh every day prior to the study (2-3        hours before injection).    -   The plethysmometer was calibrated each day prior to the study.    -   If carrageenan injection causes significant bleeding or a        hematoma on the treated foot, the animal was excluded from the        study.    -   Each paw was marked at the tibio-tarsal joint across the ankle        prior to measurements, to ensure each paw was submerged at the        same level.    -   If reading on the volume needs to be repeated, the paw had to be        dried off completely.

Statistical Analysis

The difference of the weight or the volume between right and left pawwas calculated for each animal for the analysis. Group data werepresented as means+/−SEM and p<0.05 were considered significant.Inter-group comparisons were carried out by unpaired student t test(between two groups) or one-way ANOVA followed by post hoc Bonferroni'smultiple comparisons.

Results

Certain compounds of the present invention showed significant reductionin edema 40%-71% p<0.05, when tested by this method.

Example 9 Mouse Ear Inflammatory Response to Topical Arachidonic Acid

Animals: Balb C Mice 23-28 gms, from Simonsen Labs, Gilroy, Calif.

Materials:

-   -   Arachidonic Acid, 99% pure from Porcine Liver (Sigma Aldrich)        reconstituted in acetone 2 mg/20 ul (200 mg/ml).    -   Inhalation anesthesia: Isoflurane 3% (Baxter).    -   Blood Sample tubes: Microtainer tubes w/ heparin (Becton        Dickinson).    -   TNFα Elisa assay (R&D Science).

Experimental Procedure

Test compounds, positive control (arachidonic acid only) and standard(Dexamethasone @ 0.1 mg/kg) prepared in solutions of acetone, ethanol oraqueous ethanol, were applied to both sides of the right ear with anEppendorf repipettor pipette, in a volume of 10 μl each side (20 μltotal). 30 Minutes later, 10 μl of arachidonic acid was applied to bothsides of the right ear (20 μl total). One hour after the application ofarachidonic acid, the mice were deeply anesthetized with isoflurane anda blood sample is taken via the orbital sinuses and placed inMicrotainer tubes. The animals were then euthanized by CO₂ inhalationand the right ears removed at the base. A uniform plug of ear tissue wasobtained using a 8 mm dermal punch. The earplugs were quickly weighed tothe nearest 0.1 mg and then flash frozen for TNFα determination.

Statistical Analysis:

Group data was presented as means+/−SEM and p<0.05 is consideredsignificant. Inter-group comparisons were carried out by unpairedstudent t tests (between two groups) or ANOVA (three or more groups)followed by post hoc Dunnet's test.

Results

The compounds of the present invention, such as:

-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl; and-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid;    showed significant reduction in edema (10 to 70%, p<0.05) when    tested by this method.

Example 10 High Glutamate-Induced Oxidative Stress Assay (HGOS)

This procedure was used to induce high glutamate-induced oxidativestress (HGOS) in a dopaminergic neuronal cell line. Using this assay thepotency and efficacy of test articles against HGOS neuronal cell injuryand cell death was established in a high throughput manner.

Materials

-   -   Dopaminergic neuronal cell lines    -   DMEM-No Glucose (Life Technologies Cat # 11966-025)    -   L-glutamine (Life Technologies Cat # 25030-081)    -   L-glutamic acid, monosodium salt (Sigma Cat # G5889)    -   D-glucose (Sigma Cat # G-6151)    -   10×HBSS buffer (pH 7.4) (950 ml Pyrogen-free water, 2.44 g/L        MgCl2.6H20, 3.73 g/L KCl, 59.58 g/L Hepes, 58.44 g/L NaCl, 1.36        g/L KH2PO4, 1.91 g/L CaCl2.2H2O and pH to 4.5 with HCl)    -   Cell Tracker Green fluorescent dye (Molecular Probes, Cat #        2925). Prepare a 5 μM solution in pre-warmed HBSS just prior to        use.    -   Sterile 96-well plates precoated with poly-D-lysine (Corning        Catalog # 3665)    -   96-well deep well mother plate, DyNA Block 1000 (VWR Catalog #        40002-008)

Neuronal Cells

The cells were seeded into 96-well plates at a density of 2000 per welland left to grow for 72 hours in a 33° C. incubator with 5% CO₂ in airatmosphere. The passage number of the cells for each assay experimentwere no later than p11 in order to minimize experimental variation.

Compound Preparation in Deep-Well Mother Plates

VWRBrand DyNA Block 1000, deep well mother plates (VWR Cat. # 40002-008)were used for the preparation of the test compounds.

All compounds were dissolved in DMEM-No Glu containing 1 mM glucose, 30mM glutamate and 1×Pen/Strep. DMEM-No Glu with 1 mM glucose and 1×P/Swas used as the negative control, DMEM-No Glucose with 1 mM glucose, 100M glutamate was used as a positive control and 100 μM Glutathione wasadded to the positive control as a standard. All of the procedures forthis involving the making and dilution of compounds were performed usingaseptic conditions and with minimal light.

Cell Preparation

The plates were removed from the incubator and examined under themicroscope for morphological appearance and density. Using an aseptictechnique and an 8-channel aspirator the media was carefully removedfrom the cells and replaced with 200 μl of 1×HBSS. This was done asquickly as possible to prevent the cells drying out. The plates werethen placed in the humidified 37° C. incubators of the Biomek 2000 SideLoader. Four plates were washed at a time so as to minimize the timethat the cells were sitting in 1×HBSS prior to addition of the compoundtest solution.

Experimental Setup

The Beckman Biomek workstations were used to load the compounds andcontrols from the mother plates onto the cell plates that were prewashedwith HBSS under sterile conditions. The plates were incubated in theupper HTS incubator at 37° C. in 5% CO₂ for exactly 16 hrs. Thefollowing day, using the Beckman Biomek workstations, the plates wereremoved from the incubator. Using Cell Tracker Addition, the compoundswere removed from the plates, washed once with 200 μM of pre-warmed1×HBSS and then 100 μL of 5 μM Cell Tracker Green was added to eachwell. The plates were incubated at 37° C. for 30 min to allow the dye toenter the cell and be cleaved by the esterases. After washing the cellstwice with prewarmed 1×HBSS, the plates were read with the 485excitation; 538 emission filter pair on a Fluoroskan.

Certain compounds of the present invention such as:

-   (6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethyl)-1-hydroxyurea;-   4-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-2-methyl-5-propyl-2,4-dihydro-pyrazol-3-one;-   4-[4-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-3-methyl-5-oxo-4,5-dihydro-pyrazol-1-yl]-benzoic    acid;-   5-Hydroxy-3-(6-hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-3H-benzofuran-2-one;-   Acetic acid    2,2-dimethyl-5-(3-methyl-but-2-enyl)-7,8,9,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   5-(2-Cyclohexylidene-ethyl)-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2-[3-(Benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   5-Bromo-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   5-Hydroxymethyl-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2,5-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-7,8,9,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-7,8,9,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiophen-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Isobutylsulfanyl-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester;-   5-Bromo-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,6:9,12-dimethano-9,10,11,12-tetrahydro-2,4,4-trimethylnaphtho[1,2-b]oxocan-8-ol;-   Acetic acid    2,2-dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-yl    ester,-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(2-Chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,6:9,12-Dimethano-9,10,11,12-tetrahydro-2-methylnaphtho[1,2-b]oxocan-8-ol;-   2-[3-(Benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;    and-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;    exhibited protection against HGOS cell injury and cell death with an    EC₅₀ in a range of 1 μM or less.

Example 11 Inflammation Assay Cell-ELAM Assay

Endothelial-Leukocyte Adhesion Molecule (ELAM), also known asE-selectin, was expressed on the surface of endothelial cells. In thisassay, lipopolysaccharide (LPS) and IL-1β were used to stimulate theexpression of ELAM; test agents were tested for their abilities toreduce this expression, in accordance with studies showing thatreduction of leukocyte adhesion to endothelial cell surface wasassociated with decreased cellular damage (e.g., Takada, M., Et al.,Transplantation 64: 1520-25, 1997; Steinberg, J. B., et al., J. HeartLung Trans. 13:306-313, 1994).

Endothelial cells may be selected from any of a number of sources andcultured according to methods known in the art; including, for example,coronary artery endothelial cells, human brain microvascular endothelialcells (HBMEC; Hess, D. C., et al., Neurosci. Lett. 213(1): 37-40, 1996),or lung endothelial cells. Cells were conveniently cultured in 96-wellplates. Cells were stimulated by adding a solution to each wellcontaining 10 μg/ml LPS and 100 pg/ml IL-1β for 6 hours in the presenceof test agent (specific concentrations and time may be adjusteddepending on the cell type) Treatment buffer was removed and replacedwith pre-warmed Fixing Solution® (100 μl/well) for 25 minutes at roomtemperature. Cells were then washed 3×, then incubated with BlockingBuffer (PBS+2% FBS) for 25 minutes at room temperature. Blocking Buffercontaining Monoclonal E-Selectin Antibody (1:750, Sigma Catalog #S-9555) was added to each well. Plates were sealed and stored at 4°overnight. Plates were washed 4× with 160 μL Blocking Buffer per well.Second Antibody-HRP diluted 1:5000 in Blocking Buffer was then added(100 μL/well), and plates were incubated at room temperature (protectedfrom light) for two hours. Plates were then washed 4× with BlockingBuffer before addition of 100 μL of ABTS Substrate solution at roomtemperature (Zymed, Catalog # 00-2024). Wells were allowed to developfor 35 minutes, before measurement at 402 nm in a Fluoroskan® Readerwith shake program for 10 seconds. Positive results were recorded as adecrease in ELAM concentration in tested wells, as compared to controlwells.

Certain compounds of this invention such as:

-   2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-[3-(Benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,6:9,12-Dimethano-9,10,11,12-tetrahydro-2-methylnaphtho[1,2-b]oxocan-8-ol;-   2-(2-Chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-butyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   5-Bromo-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Isobutylsulfanyl-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    and-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    when tested as described above showed activity at an EC₅₀ of 30 μM    or less.

Example 12 Inhibition of LTB₄ Production in Blood Materials

Fresh heparinized whole blood.NDGA, control 5-Lipoxygenase inhibitor.A23187, calcium ionophore (Sigma C-7522)

DMSO Methanol Ethanol EIA, ELISA Kit (Cayman Chemical Co., Ann Arbor,Mich., USA) Procedure

The blood was collected in heparinized tubes or syringes to preventclotting. Heparin was at 20 USP U/ml. About 3-5 ml of blood wascollected per rat and about 0.5-0.75 ml per mouse. This assay required200 μl/data point, therefore a 96 well plate requires 20 ml.

The Calcium ionophore A23187 was prepared as a 10 mM stock solution inDMSO and the test agent is prepared in DMSO so that <1 μl DMSO will beadded to the well

Assay:

200 μl blood per well were added in a round bottom 96 well clear plastic96 well plate followed by the test agent. After 10 minutes incubation at37° C., 5 μl of Calcium ionophore A23187 for a final concentration of 20μM were added and mixed, and further incubated for 30 min. After 1000×gcentrifugation to pellet blood cells, 20 μl plasma was carefullyremoved.

20 μl plasma were added to 80 μl methanol to precipitate protein andmixed. This was further incubated 10 min. at room temperature andcentrifuged at 1000×g to pellet protein.

Following manufacturer's instructions, the commercially available EIAkit was used to subsequently measure the LTB₄ production in the samples.The LTB₄ levels produced in the vehicle control sample were thencompared to those in which the test compound had been added. From this apercent inhibition of LTB.sub.4 production by each concentration of testcompound was calculated and the IC₅₀ for inhibition of LTB₄ productionfor each test compound was determined.

Certain compounds of this invention such as:

-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   4-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-2-methyl-5-propyl-2,4-dihydro-pyrazol-3-one;-   2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   [3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonic    acid dimethyl ester;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Isobutylsulfanyl-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiophen-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2,5-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    and-   5-Bromo-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    when tested as described above provided protection against LTB₄ at    an EC₅₀ of 10 μM or less.

Example 13 LTB₄-Cell Assay

This procedure was used for measuring the release of the leukotrieneLTB4 from a neutrophil cell line using a competitive ELISA technique.

Materials and Equipments Materials for Cell Preparation and Experiment

-   -   MPRO cell line (ATCC, Catalog # CRL-11422)    -   Calcium ionophore (A23187) (Sigma, Catalog # C7522)    -   Nordihydroguaiaretic acid (NDGA) (BioMol, Catalog # EI101-0001)    -   Retinoic Acid (all-trans) (ATRA) (Sigma, Catalog # 95152)    -   Sterile, tissue-culture treated 96-well plates (Corning, Catalog        # 3614)

Materials for LTB4 ELISA

-   -   Precoated (Mouse Anti-Rabbit IgG) EIA 96 Well Strip Plates        (Cayman, Catalog # 400004)    -   Leukotriene B4 AChE Tracer (Cayman Catalog # 420110)    -   Leukotriene B4 EIA Antiserum (Cayman Catalog # 420112)    -   Ellman's Reagent (Cayman Catalog # 400050)    -   EIA Buffer Concentrate (10×) (Cayman Catalog # 400060)    -   Wash Buffer Concentrate (400×) (Cayman Catalog # 400062)    -   Plastic plate covers (Cayman Catalog # 400012)

Procedure

A mouse promyelocytic cell line (MPRO) was used in this assay. Thesecells are committed immature neutrophils that can be differentiated intomature neutrophils by treatment with 10 μM all-trans retinoic acid for72 hours

Following 72 hours of differentiation, cells were stimulated with 1 μMof a calcium ionophore (A23187) in the presence or absence of testcompound or vehicle for 1 hour at 37° C. After this time, supernatantwas removed from the cells and the LTB4 levels were determined followingmanufacturer's instructions, using a Leukotriene B4 EIA kit from Cayman(Cat # 520111)

The negative controls were media samples from differentiated butunstimulated cells.

The compounds were screened at 5 concentrations in quadruplicatestarting at 10 μM

Following the procedure described above certain compounds of the presentinvention, such as:

-   2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(□yridine-4-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Isobutylsulfanyl-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiophen-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiazol-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-7,8,9,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2,5-Trimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    and-   Dimethylamino-acetic acid    2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-yl    ester.    were considered to be active if they exhibited inhibition of LTB4    with an EC₅₀ in a range of 5 μM or less.

Example 14 5-Lypoxygenase Enzyme Assay

This procedure was used for measuring the enzymatic activity of humanrecombinant 5-lipoxygenase using a calorimetric method based on theferric oxidation of xylenol orange.

Materials

-   -   96 well flat bottom microfilter plates (VWR, Catalog # 62402-933        9295)    -   Lipoxygenase screening assay buffer (Cayman, Catalog # 760710)    -   Human recombinant 5-lipoxygenase (Cayman, Catalog # 60402)    -   Arachidonic Acid (Sigma, Catalog # A3555)    -   Xylenol orange tetrasodium salt (Aldrich, Catalog # 227854)    -   Iron (II) sulfate heptahydrate (Sigma, Catalog # F7002)    -   Sulfuric acid (95-98%) [18M]    -   Methanol

Procedure

Human recombinant 5-lipoxygenase (Cayman Cat # 60402) was used in thisassay. The test compound and/or vehicle was added to 0.5U 5-lipoxygenasein 50 mM Tris-HCl buffer, pH 7.4. The reaction was initiated by additionof 70 μM arachidonic acid in Tris-HCl buffer, pH 7.4, and terminatedafter a 10 minute incubation at room temperature by addition of FOXreagent (25 mM sulphuric acid, 100 μM xylenol orange, 100M iron (II)sulphate, methanol:water 9:1). The yellow color of acidified xylenolorange was converted to a blue color by the lipid hydroperoxide-mediatedoxidation of Fe²⁺ ions and the interaction of the resulting Fe³⁺ ionswith the dye. The complex was allowed to form during a 1 hour incubationat room temperature with shaking. Absorbance of the Fe³⁺ complex wasthen measured at 620 nM using a spectrophotometer.

Negative controls contained enzyme during the incubation step butsubstrate was not added until after the FOX reagent.

Compounds were screened at 5 concentrations in triplicate starting at 10μM

Certain compounds of this invention such as

-   2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(□yridine-4-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,10-tetrahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   10-Methoxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid methyl ester;-   2,2-Dimethyl-7,8,9,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2-(3-Chloro-propyl)-2,5-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   5-Bromo-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    and-   3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionic    acid benzyl ester;    when tested as described above provided protection against    5-Lipoxygenase with an IC₅₀ of 5 μM or less.

Example 15 12/15-Lipoxygenase Enzyme Assay

This procedure was used for measuring the enzymatic activity of porcineleukocyte 12/15-lipoxygenase using a calorimetric method based on theferric oxidation of xylenol orange.

Materials

-   -   96 well flat bottom microfilter plates (VWR, Catalog # 62402-933        9295)    -   Lipoxygenase screening assay buffer (Cayman, Catalog # 760710)    -   Porcine leukocyte 12/15-lipoxygenase (Cayman, Catalog # 60300)    -   Arachidonic Acid (Sigma, Catalog # A3555)    -   Xylenol orange tetrasodium salt (Aldrich, Catalog # 227854)    -   Iron (II) sulfate heptahydrate (Sigma, Catalog # F7002)    -   Sulfuric acid (95-98%) [18M]    -   Methanol

Procedure

Porcine Leukocyte 12/15-lipoxygenase (Cayman Cat # 60300) was used inthis assay. Test compound and/or vehicle was added to 1.3U12/15-lipoxygenase in 50 mM Tris-HCl buffer, pH 7.4. The reaction wasinitiated by addition of 70 μM arachidonic acid in Tris-HCl buffer, pH7.4, and terminated after a 10 minute incubation at room temperature byaddition of FOX reagent (25 mM sulphuric acid, 100 μM xylenol orange,100 μM iron (II) sulphate, methanol:water 9:1). The yellow color ofacidified xylenol orange was converted to a blue color by the lipidhydroperoxide-mediated oxidation of Fe²⁺ ions and the interaction of theresulting Fe³⁺ ions with the dye. The complex was allowed to form duringa 1 hour incubation at room temperature with shaking. Absorbance of theFe³⁺ complex was then measured at 620 nM using a spectrophotometer.

Negative controls contained enzyme during the incubation step butsubstrate was not added until after the FOX reagent.

Compounds are screened at 5 concentrations in triplicate starting at 10μM

Certain compounds of the present invention such as:

-   2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-[3-(□yridine-4-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-(3-Isobutylsulfanyl-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiophen-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2-Methyl-2-thiazol-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;-   2,2-Dimethyl-7,8,9,10-tetrahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    and-   2,2,5-Trimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;    were considered to be active when they exhibited inhibition of    12/15-Lipoxygenase with an IC₅₀ of 10 μM or less.

Example 163-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicAcid Methyl Ester

Step 1:

To a solution of ethyl 2-ketopentanoate (10 mL, 71 mmol) in 100 mL ofTHF at −78° C. was added dropwise a solution of 1.0 M vinyl magnesiumbromide (80 mL, 80 mmol). After completion of the addition, the mixturewas stirred for two more hours, the cooling bath was removed and themixture was gradually warmed to ambient temperature. After the removalof the most of the solvent, the residue was poured to 2N HCl aqueoussolution, extracted with ethyl acetate and dried over MgSO₄.Purification by lash column chromatography on silica gel with DCM aselute afforded 4.80 grams of 5-methyl-5-vinyl-dihydro-furan-2-one.¹H-NMR (300 MHz, CDCl₃) δ (ppm): 5.824-5.917 (dd, 1H); 5.29 (d, 1H);5.13 (d, 1H); 2.50-2.56 m, (2H); 2.07-2.18 (m, 2H); 1.48 (s, 3H).

Step 2:

A mixture of 1,2,3,4-tetrahydro-1,4-methano-naphthalene-5,8-diol (1.34g, 7.94 mmol), 1.0 mL (3.97 mmol) of BF₃.Et₂O and 25 mL of dioxane washeated up to 110° C. under a nitrogen atmosphere. A solution of5-methyl-5-vinyl-dihydro-furan-2-one (1.12 mg, 8.6 mmol) in 24 ml ofdioxane was added slowly to the mixture. The addition was completedafter two hours and the mixture was continued to reflex for additional 3hours. After cooling down to room temperature, the mixture was poured towater, extracted with ethyl acetate, washed with water and dried overMgSO₄. After the removal of solvents, the residue was mixed withmethanol/HCl and let stand overnight at room temperature. Then thesolvents were removed and the residue was purified via flash columnchromatography on silica gel (1:3 EtOAc/Hex) twice to give 1.30 g of3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.29 (s, 1H), 4.70(s, 1H), 3.68 (s, 3H), 3.52-3.46 (d, 2H), 2.69-2.55 (m, 2H), 2.52-2.47(m, 2H), 2.00-1.64 (m, 8H), 1.43-1.15 (m, 5H). MS (ESI): 317 (M+H, 70%),339 (M+Na⁺, 100%).

Example 173-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicAcid

1.0 gram (3.16 mmol) of3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester, prepared as described in Example 13, was mixed with20 mL of 2N NaOH and 20 mL of dioxane, the mixture was stirred for 5hours and was acidified with dilute HCl. Water and ethyl acetate wereadded and the organic layer was separated, washed and dried over MgSO₄.After the removal of solvents, the residue was further purified by flashcolumn chromatography on silica gel (3:2 ethyl acetate/hexane) to afford700 mg of3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.29 (s, 1H), 4.70 (s, 1H),3.52-3.46 (d, 2H), 2.69-2.55 (m, 2H), 2.52-2.47 (m, 2H), 2.00-1.64 (m,8H), 1.43-1.15 (m, 5H); MS: 302 (M+H⁺, 73%), 325 (M+Na⁺, 100%)

Example 183-(6-Methoxymethoxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicAcid Methyl Ester

To a solution of3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester from Example 13 (400 mg, 1.27 mmol) and diisopropylethylamine (DIPEA) (0.90 ml, 5 mmol) in 4 mL of DCM was slowly addedchloromethyl methyl ether (0.30 mL, 3.8 mmol) at room temperature. Thereaction was left over night. After the completion of reaction shown byTLC, the mixture was poured into brine and the organic layer wasseparated and dried over MgSO₄. After the removal of solvents theresidue was purified through flash column chromatography on silica gel(25% of ethyl acetate in hexane) to afford 340 mg of3-(6-methoxymethoxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester. ¹H-NMR (300 MHz, CDCl₃) δ (ppm)): 6.25 (s, 1H), 4.29(s, 1H), 3.63 (s, 3H), 3.48 (s, 1H), 3.40 (s, 1H), 2.66-2.64 (m, 2H),2.47-2.43 (m, 2H), 1.83-1.50, (m, 12H) 1.18 (s, 3H).

Example 192-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol

To the mixture of lithium aluminum hydride (LiAlH₄, 713 mg, 18 mmol) in40 mL of dried THF under nitrogen was added drop-wise a solution of theMOM-protected methyl ester from Example 15 (2.70 g, 7.5 mmol), and thereaction mixture was stirred overnight. Ethyl acetate was slowly addedto the mixture to destroy the excess of LiAlH₄. The mixture was filteredand the solution was washed with water and dried over MgSO4. The solventwas removed and the residue was purified via flash column chromatographyon silica gel (2:3 EtOAc/Hex) to afford 2.15 grams of 6-methoxymethoxy3-hydroxy-propyl-alcohol chroman derivative.

¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.50 (s, 1H), 5.02 (s, 2H), 3.60 (m,2H), 3.44 (s, 2H), 3.41 (s, 3H), 2.83 (s, 1H), 2.64 (m, 2H), 1.80-1.50(m, 12H), 1.25 (s, 3H); MS: 333 (M+H⁺, 10%), 355 (M+Na⁺, 100%)

A mixture of 200 mg of the above synthesized MOM-protected alcohol, 6.5mL of methanol and 6 drops of concentrated HCl was stirred overnight.The solvent was removed carefully and the residue was partitioned withethyl acetate and water. The ester layer was separated and washed withwater and dried over Na₂SO₄. Removal of solvent followed by flash columnchromatography on silica gel (2:3 ethyl acetate/hexane) afforded 160 mgof2-(3-hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol.

¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.29 (s, 1H), 4.35 (s, 1H), 3.56-3.70(m, 2H), 3.50 (s, 1H), 3.40 (s, 1H), 2.66-2.72 (m, 2H), 1.52-1.90 (m,12H), 1.23-1.28 (d, 3H)

MS: 289 (M+H⁺, 80%), 311 (M+Na⁺, 75%)

Example 20[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicAcid Dimethyl Ester

Step 1:

To a mixture of 6-methoxymethoxy 3-hydroxy-propyl-alcohol chromanderivative (287 mg, 0.86 mmol), prepared as described above,triethylamine (18.5 mL, 1.3 mmol) and 5 ml of DCM, was added methanesulfonyl chloride (8 mL, 1.04 mmol). After 5 hours of stirring themixture was poured into water, extracted with ethyl acetate, and driedover MgSO₄. Removal of solvent afforded 300 mg of the mesylatederivative. This product is pure enough to go directly to next step.

Step 2:

A mixture of 800 mg of methanesulfonate derivative from Step 1, 1.0 gramof sodium iodide and 20 mL of acetone was stirred overnight. The solventwas removed, and a mixture of ethyl acetate and water was added to theresidue, the organic layer was separated and dried. The solvents wereremoved and the residue was purified via flash column chromatography onsilica gel using 15:1 hexane/ethyl acetate to afford 520 mg of theiodide derivative.

Step 3:

A mixture of 650 mg of the iodide derivative from Step 2 and 5 mL oftrimethyl phosphite was refluxed for 4 hours followed by distillation ofthe excess of the starting materials. The mixture was cooled to roomtemperature, and was purified by flash column chromatography on silicagel (5% MeOH/DCM) to afford 610 mg of[3-(6-methoxymethoxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicacid methyl ester. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.38 (s, 1H), 5.11(s, 2H), 3.72-3.82 (m, 6H) 3.53 (s, 3H), 2.62-2.68 (m, 4H), 1.70-1.84(m, 12H), 1.22-1.28 (d, 3H); MS: 425 (M+H⁺, 65%), 403 (M+Na⁺, 100%).

Step 4:

190 mg of 6-methoxymethoxy-dimethyl phosphonate derivative from Step 3was treated with a mixture of 20 mL of MeOH and 20 drops of concentrateHCl for overnight. After removal of most of the solvent, ethyl acetatewas added and then washed with water, and dried over MgSO₄. After theremoval of solvents the residue was purified by flash columnchromatography on silica gel (10% MeOH I DCM) to afford 90 mg of[3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicacid dimethyl ester. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.38 (s, 1H), 5.32(s, 1H), 3.72-3.82 (m, 6H) 3.50-3.54 (m, 2H), 2.62-2.68 (m, 4H),1.70-1.84 (m, 12H), 1.22-1.28 (s, 3H) MS: 381 (M+H⁺, 100%), 403 (M+Na⁺,36%)

Example 21[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicAcid

A mixture of 90 mg of dimethyl phosphonate, prepared as described above,0.3 mL of trimethylsilylbromide in 6 mL of DCM was stirred at roomtemperature for 2 hours. The solvent and excess of trimethylsilylbromidewas evaporated and the residue was mixed with 3 mL of 1:3 MeOH/H2O andwas then dried overnight to afford[3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicacid as a white solid. ¹H-(NMR, CD₃OD) δ (ppm): 6.05 (s, H), 4.87 (s,2H), 3.27-3.29 (d, 2H), 3.12-3.13 (m, 1H), 2.48 (m, 2H), 1.70-1.84 (m,12H), 1.22-1.28 (d, 3H). MS: 353 (M+H⁺, 100%).

Example 222-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol

Step 1:

A mixture of 50 mg methanesulfonate prepared as described above, 86 mgof LiBr and 1.8 ml of acetone was stirred overnight. After the removalof solvent the residue was purified via flash column chromatography onsilica gel (1:5 EtOAc/Hex) to afford 29 mg of the6-methoxymethoxy-propylbromide derivative. ¹H-NMR (300 MHz, CDCl₃) δ(ppm): 6.60 (s, 1H), 5.10 (s, 2H), 3.60 (m, 2H), 3.54 (m, 3H), 2.64 (m,4H), 1.80-1.50 (m, 12H), 1.25 (s, 3H)

Step 2:

A mixture of bromide from Step 1 (190 mg, 0.48 mmol), 76 mg (0.647 mmol)of thiazole-2-thiol, 200 mg (1.45 mmol) of K₂CO₃, small amount of NaIand 5 ml of acetone was stirred overnight. The mixture was filtered andthe filtrate was concentrated by evaporation. The residue was purifiedvia flash column chromatography on silica gel (1:3 EtOAc/Hex) to afford200 mg of the 6-methoxymethoxy-2-[3-(thiazol-2-ylsulfanyl)-propylchroman derivative. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.71 (s, 1H), 7.18(s, 1H), 6.56 (s, 1H), 5.12 (s, 2H), 3.54 (s, 3H), 3.10-3.28 (m, 2H),2.65-2.77 (m, 2H), 1.63-2.01 (m, 8H), 1.58 (s, 3H), 1.18-1.32 (m, 6H)

Step 3:

200 mg of thiazole from Step 2 was treated with a mixture of 20 mL ofMeOH and 20 drops of concentrate HCl and stirred overnight. Afterremoval of the solvent, ethyl acetate was added and the mixture waswashed with water and dried over MgSO₄ to give 140 mg of2-methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol.¹H-NMR (300M Hz, CDCl₃) δ (ppm): 7.69 (s, 1H), 7.23 (s, 1H), 6.32 (s,1H), 4.19 (s, 1H), 3.46-3.54, (d, 2H), 3.23-3.26 (m, 2H), 2.69-2.72 (m,2H), 2.07-1.77 (m, 12H), 1.31-1.24 (m, 6H); MS: 388 (M+H⁺, 100%).

Example 234-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-butane-1-sulfonicAcid Dimethylamide

Step 1:

To a solution of N,N-dimethylmethanesulfonamide (369 mg, 3 mmol) in 4 mlof THF at −78° C. was added a solution of 2.5 M n-BuLi in hexane (0.8mL, 2 mmol). The mixture was stirred at this temperature for one hourand then a solution of the methanesulfonate prepared as described above(205 mg, 0.5 mmol) in 2 mL of THF was added and the mixture wasgradually warmed to room temperature and stirred over night. The mixturewas poured into water, extracted with ethyl acetate, and dried overMgSO₄. After the removal of solvent, the residue was purified via flashcolumn chromatography on silica gel with 1:2 ethyl acetate/hexane toafford 75 mg of the bicyclic 6-methoxymethoxy-2-butyldimethylsulfonamidechroman derivative. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.30 (s, 1H),3.50-3.54 (m, 2H), 2.88-3.00 (m, 1H), 2.60-2.70 (m, 4H), 1.25-1.84 (m,12H), 1.22-1.28 (d, 3H); MS: 394 (M+H⁺, 100%), 416 (M+Na⁺, 70%)

Step 2

A mixture of 70 mg of 6-methoxymethoxy-2-butyldimethylsulfonamidechroman derivative from Step 1, 8 ml of methanol and 0.1 ml of conc. HClwas stirred overnight. The solvent was removed and the residue waspartitioned with ethyl acetate and water. The ester layer was separatedand washed with water and dried over Na₂SO₄. Removal of the solventfollowed by flash column chromatography on silica gel (3:2 EtOAc/hexane)afforded 31 mg of4-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-butane-1-sulfonicacid dimethylamide ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.29 (s, 1H), 3.54(s, 1H), 3.45 (s, 1H), 2.88-2.99 (m, 1H), 2.80 (m, 6H), 2.60-2.72 (m,4H), 1.50-1.88 (m, 12H), 1.23-1.28 (d, 3H), 1.22-1.31 (2H); MS: 394(M+H⁺, 100%), 416 (M+Na⁺, 70%)

Example 242-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol

Step 1:

To a solution of 5-chloro-pentane 2-one (13.4 mL, 100 mmol) in 100 ml ofTHF at −78° C. was added dropwise a solution of 1M vinyl magnesiumbromide (120 mL, 120 mmol), the mixture was stirred for two hours, andthe cooling bath was removed to let the mixture gradually warm to roomtemperature. After the removal of the solvent, the residue was poured to2N HCl, extracted with ethyl acetate and dried over MgSO₄. Purificationby flash column chromatography on silica gel with DCM as elute afforded4.80 grams of 6-chloro-3-methyl-hex-1-en-3-ol. ¹H-NMR (300 MHz, CDCl₃) δ(ppm): 5.86-5.90 (dd, 1H); 5.29 (d, 1H), 5.13 (d, 1H), 3.50-3.59 (t,3H); 1.87-1.83 2.18 (m, 2H); 1.63-1.69 (m, 2H), 1.31 (s, 3H)

Step 2

A mixture of 1,2,3,4-tetrahydro-1,4-methano-naphthalene-5,8-diol (1.34g, 794 mmol), 1.0 mL (3.97 mmol) of BF₃.Et₂O and 25 ml of dioxane washeated up to 110° C. under a N₂ atmosphere. A solution of6-chloro-3-methyl-hex-1-en-3-ol (1.12 g, 8.6 mmol) in 24 ml of dioxanewas added slowly to the mixture. The addition was completed after twohours and the mixture was continued to reflux for an additional 3 hours.After cooling down to room temperature, the mixture was poured to water,extracted with ethyl acetate, washed with water and dried over MgSO₄.After removal of the solvents, the residue was mixed with methanol/HCland let stand overnight at room temperature. After the removal of thesolvents the residue was purified via flash column chromatography onsilica gel (1:3 EtOAc/Hex) twice to give 1.30 g of2-(3-chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol.¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.39 (s, 1H), 4.27 (s, 1H), 3.63-3.47(m, 4H), 2.73-2.70 (m, 2H), 2.00-1.47 (m, 8H), 1.43-1.15 (m, 4H); MS:307 (M+H⁺, 100%).

Example 252-[3-(Benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol

Step 1:

A mixture of 612 mg of the 3-chloropropyl chroman derivative prepared asdescribed above (2 mmol), 1.5 gram of sodium iodide (10 mmol) and 8 mlof acetone was refluxed for 6 hours. The mixture was cooled down to roomtemperature and the solvent was removed by evaporation. The residue wasdissolved in DCM, washed with water, and the organic layer was separatedand dried over MgSO₄. Removal of solvent afforded the crude iodide whichwas used directly in the next step.

Step 2:

A mixture of 100 mg of 3-iodopropyl chroman derivative from Step 1 (0.25mmol), 66 mg of benzothiazole-2-thiol (0.39 mmol), and 55 mg of K₂CO₃(0.39 mmol) in 1.5 ml of DMF was stirred overnight. The mixture waspoured into water and extracted with DCM. Purification by flash columnchromatography on silica gel (3:1 Hexane/EtOAc) of the residue gave 55mg of pure2-[3-(benzothiazol-2-ylsulfanyl)-propyl]-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol.¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.86 (m, 1H), 7.78 (m, 1H), 7.41 (m,1H), 7.26 (m, 1H), 6.33 (s, 1H), 4.23 (s, 1H), 3.50-3.42 (m, 2H),3.38-3.25 (m, 2H), 2.80 (m, 2H), 2.07-1.77 (m, 12H), 1.31-1.24 (m, 6H);MS: 437 (M+H⁺, 100%).

Example 261-{3-[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propylsulfanyl]-2-methyl-propionyl}-pyrrolidine-2-carboxylicAcid

A mixture of 200 mg of iodide (0.50 mmol), 163 mg1-(3-mercapto-2-methyl-propionyl)-pyrrolidine-2-carboxylic acid (0.75mmol), 97 mg of K₂CO₃ (0.70 mmol) in 2.0 ml of DMF was stirredovernight. The mixture was poured into water and extracted with DCM.Purification by flash column chromatography on silica gel (4% ofMeOH/DCM) of the residue gave 65 mg of1-{3-[3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propylsulfanyl]-2-methyl-propionyl}-pyrrolidine-2-carboxylicacid. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.31 (s, 1H), 4.65 (m, 1H), 4.55(m, 2H), 4.19 (s, 2H), 3.50-3.42 (m, 2H), 2.90 (m, 1H), 2.80-2.67 (m,5H), 2.43-2.35 (m, 2H), 2.07-1.77 (m, 12H), 1.31-1.24 (m, 10H); MS: 488(M+H⁺, 100%), 510 (M+Na⁺, 65%).

Example 273-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionicAcid Methyl Ester

Step 1:

A sealed reaction vessel containing quinone (700 mg, 6.48 mmol) andcycloheptadiene (731 mg, 7.76 mmol) was heated in a microwave reactor(Personal Chemistry) to 170° C. for 15 min. The solvent was removedunder reduced pressure to afford a dark brown solid containing desiredproduct, hydroquinone and impurities. The crude products from 17 runs ofsuch reaction (11.9 g, 0.11 mol) were combined and chromatographed toafford a brown solid (8 g) containing the desired compound and quinone.To this material in 150 mL dry acetone was added 7 g of K₂CO₃ and themixture was heated to reflux for 2 h and cooled and concentrated underreduced pressure. The crude product was purified by columnchromatography on silica gel (hexanes/EtOAc=5:1) to afford of5,8-dihydro-5,8-propano-naphthalene-1,4-diol as a light brown solid (4.6g). MS (ESI) m/z: 203 (M+H⁺, 100%)

Step 2:

To a solution of the hydroquinone prepared in Step 1 in 100 mL ethanolwas added Pd/C (250 mg) charged with hydrogen. The reaction was stirredunder hydrogen for 5 h and the solid was removed through filtration. Thesolvent was removed and the residue was chromatographed to afford5,6,7,8-tetrahydro-5,8-propano-naphthalene-1,4-diol as a light brownsolid (3.9 g). ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.51 (s, 2H), 3.33 (bs,2H), 1.96-1.89 (m, 2H), 1.70-1.57 9 (m, 6H), 1.18 (m, 2H);

MS (ESI) m/z: 205 (M+H⁺, 100%).

Step 3:

To a solution of 5,6,7,8-tetrahydro-5,8-propano-naphthalene-1,4-diolfrom Step 2 (3.7 g, 18.1 mmol) in 80 mL of dioxane was added BF₃.Et₂O(7.7 g, 54.5 mmol). The mixture was heated to reflux and vinyl lactone(2.28 g, 18.1 mmol, in 20 mL dioxane) was added over a period of 60 min.The reaction was stirred at reflux for 16 h and cooled. It was quenchedon to ice (300 g) and extracted with DCM (3×120 mL). The combinedorganic layers were dried over Na₂SO₄, concentrated and dried under highvacuum. To this crude material in 250 mL MeOH was added 30 mg oftoluenesulfonic acid. The mixture was heated to reflux for 15 h andcooled. After solvent removal the residue was chromatographed(hex/EtOAc=7:1) to afford3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester as a green sticky oil (1.4 g). ¹H-NMR (300 MHz, CDCl₃)δ (ppm): 6.41 (s, 1H), 3.70 (s, 3H), 3.53 (m, 1H), 3.41 (m, 1H), 2.72(m, 2H), 2.52 (m, 2H), 2.09-1.62 (12H), 1.29-1.24 (5H); ¹³C-NMR δ(ppm):174.5, 144.0, 142.9, 132.8, 128.6, 118.5, 112.4, 74.1, 51.7, 34.5, 33.9,31.9, 29.5, 28.6, 26.3, 26.0, 24.0, 23.4, 22.7, 22.3; MS (ESI) m/z: 345(M+H+, 100%)

Example 282-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-4-ol

To a solution of3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester (212 mg, 0.61 mmol) in 50 mL dry THF was added LiAlH₄in 8 portions over a period of 20 min. The reaction was stirred for 2 hand quenched onto ice (80 g). The mixture was extracted with EtOAc (3×50mL) and combined organic layers were dried over Na₂SO₄ and concentrated.The crude product was chromatographed (hexane/EtOAc=6:1) to yield2-(3-hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-6-olas a light yellow oil (169 mg). ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 6.39(s, 1H), 5.78 (s, 1H), 3.69 (m, 2H), 3.54 (m, 1H), 3.40 (m, 1H), 2.69(m, 2H), 2.45 (ws, 1H), 1.90-1.60 (m, 14H), 1.26 (m, 5H); ¹³C-NMR δ(ppm): 144.3, 142.9, 132.7, 128.9, 118.6, 112.6, 75.0, 63.3, 36.1, 35.7,32.1, 32.0, 31.9, 29.4, 28.7, 26.8, 26.4, 26.1, 24.2, 23.6, 22.8, 22.4;MS (ESI) m/z: 317 (M+H⁺, 100%).

Example 293-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionicAcid

To a solution of3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester (235 mg, 0.68 mmol) in 5 mL of dioxane was added asolution of NaOH (41 mg, 1.02 mmol) in 400 mL water. The resultingsuspension was vigorously stirred for 15 h and quenched by adding 50 mLof saturated NaH₂PO₄ solution. The mixture was extracted with EtOAc(3×50 mL) and the organic layers were dried over Na₂SO₄ andconcentrated. The crude product was purified by chromatography on silicagel to afford3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionicacid as a white sticky solid (184 mg). ¹H-NMR (CDCl₃, 300 MHz) δ (ppm):7.71 (ws, 1H), 6.41 (s, 1H), 3.54 (m, 1H), 3.38 (m, 1H), 2.73 (m, 2H),2.56 (m, 2H), 2.10-1.62 (m, 12H), 0.95 (m, 5H); ¹³C-NMR δ (ppm): 180.1,144.0, 142.9, 132.8, 128.9, 118.5, 112.7, 74.0, 34.48, 34.0, 32.0, 31.9,31.8, 29.5, 28.7, 28.6, 26.4, 26.3, 26.0, 23.9, 23.3, 22.7, 22.3; MS(ESI) m/z: 331 (M+H⁺, 100%).

Example 302-(2-Chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol

Step 1:

To a solution of 4-chloro-butanone-2 (2.65 g, 25 mmol) in 25 ml of THFat −20° C. was added dropwise a solution of 1M vinyl magnesium bromide(40 ml, 40 mmol). After the completion of addition the mixture wasstirred for one more hours at room temperature. To the mixture was added10 ml of water at 0° C. with stirring. The mixture was dried over MgSO₄.After the removal of the solvent, the residue was purified via flashcolumn chromatography on silica gel with 1:3 ethyl acetate and hexane aselute to afford 800 mg of 5-chloro-3-methyl-pent-1-en-3-ol. ¹H-NMR (300MHz, CDCl₃) δ (ppm): 5.86-5.90 (dd, 1H); 5.29 (d, 1H), 5.13 (d, 1H),3.50-3.59 (t, 3H); 1.87-1.83 2.18 (m, 2H); 1.63-1.69 (m, 2H), 1.31 (s,3H)

Step 2:

A mixture of, 1,2,3,4-tetrahydro-1,4-methano-naphthalene-5,8-diol (1.34g, 794 mmol), 11.0 ml 3.97 mmol) of BF₃.Et₂O and 25 ml of dioxane washeated up to 110° C. under a nitrogen atmosphere. A solution of5-chloro-3-methyl-pent-1-en-3-ol (1.12 g, 8.6 mmol) in 24 ml of dioxanewas added slowly to the mixture. The addition was completed after twohours and the mixture was continued to reflux for an additional 3 hours.After cooling down to room temperature, the mixture was poured to water,extracted with ethyl acetate, washed with water and dried over MgSO₄.After the removal of solvents, the residue was mixed with methanol/HCland let stirred overnight at room temperature. After the removal of thesolvents the residue was purified via flash column chromatography onsilica gel (1:3 EtOAc/Hex) twice to give 1.30 g of2-(2-chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol.¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.39 (s, 1H), 4.27 (s, 1H), 3.63-3.47(m, 4H), 2.73-2.70 (m, 2H), 2.00-1.47 (m, 8H), 1.43-1.15 (m, 4H); MS:307 (M+H⁺, 100%).

Similarly, substituting in Step 1, 4-chloro-butanone-2 with5-chloro-pentan-2-one, 6-chloro-3-methyl-hex-1-en-3-ol was produced(¹H-NMR (300 MHz, CDCl₃): 5.86-5.90 (dd, 1H); 5.29 (d, 1H), 5.13 (d,1H), 3.50-3.59 (t, 3H); 1.87-1.83 2.18 (m, 2H); 1.63-1.69 (m, 2H), 1.31(s, 3H)), and cyclization with 3′,6′-dihydrobenzo-norbornane gave of2-(3-chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol:¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.39 (s, 1H), 4.27 (s, 1H), 3.63-3.47(m, 4H), 2.73-2.70 (m, 2H), 2.00-1.47 (m, 8H), 1.43-1.15 (m, 4H); MS:307 (M+H⁺, 100%)

Similarly, substituting in Step 1, 4-chloro-butanone-2 with1-thiophen-2-yl-ethanone, followed by cyclization with3′,6′-dihydrobenzo-norbornane gave2-methyl-2-thiophen-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol,¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.28 (m, 1H), 7.00 (m, 2H), 4.20 (m,1H), 3.51 (m, 2H), 2.90-2.78 (m, 2H), 2.43-2.24 (m, 2H), 1.96-1.80 (m,3H), 1.77-1.74 (m, 4H), 1.38-1.20 (m, 3H); MS: 313 (M+H⁺, 38%), 335(M+Na⁺, 100%)

Similarly, substituting in Step 1, 4-chloro-butanone-2 with1-thiazol-2-yl-ethanone, followed by cyclization with3′,6′-dihydrobenzo-norbornane gave2-methyl-2-thiazol-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2-H-benzo[h]chromen-6-ol,¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.74 (dd, 1H), 7.19 (dd, 1H), 6.39 (s,1H), 4.67 (s, 1H), 3.53-3.47 (m, 4H), 2.26 (s, 3H), 1.91-1.88 (m, 3H),1.70 (m, 1H), 1.52 (m, 1H), 1.31-1.21 (m, 3H); MS: 314 (M+H⁺, 100%)

Example 312-Methyl-2-[3-(pyridin-4-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol

A mixture of 100 mg of iodide (0.25 mmol), 66 mg of 4-mercapto-pyridine(0.39 mmol), 55 mg of K₂CO₃ (0.39 mmol) in 1.5 mL of DMF was stirredovernight. The mixture was dumped to water and extracted with DCM.Purification flash column chromatography on silica gel (3:1Hexane/EtOAc) of the residue gave 55 mg of2-methyl-2-[3-(pyridin-4-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol.¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.86 (m, 1H), 7.78 (m, 1H), 7.41 (m,1H), 7.26 (m, 1H), 6.33 (s, 1H), 4.23 (s, 1H), 3.50-3.42 (m, 2H),3.38-3.25 (m, 2H), 2.80 (m, 2H), 2.07-1.77 (m, 12H), 1.31-1.24 (m, 6H);MS: 382 (M+H⁺, 100%)

Similarly substituting 4-mercapto-pyridine with 2-methyl-propane-1-thiolgave2-(3-isobutylsulfanyl-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.34 (s, 1H), 4.22 (s, 1H), 3.60-3.44(m, 2H), 2.73-2.68 (m, 2H), 2.60-2.50 (m, 2H), 2.42-2.38 (m, 2H),1.88-1.40 (m, 6H), 1.29-1.26 (m, 10H), 0.95-0.84, (m, 6H); MS: 361(M+H⁺, 100%).

Example 323-[6-hydroxy-2-methyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl]-propionicacid

A solution of3-(6-hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid, (2.39 mmol) in 1M aq. NaOH (4.8 mL, 4.8 mmol) was cooled to 0° C.,and treated with prenyl bromide (0.277 mL, 2.39 mmol). Followingstirring for 7 h at ambient temperature the reaction mixture was madeslightly acidic with 0.5M HCl and shaken with EtOAc. The organic phasewas evaporated yielding 1. The mixture was subjected to columnchromatography on silica gel (SiO₂:hexane:EtOAc, 85:15 v/v elutingyielded3-[6-hydroxy-2-methyl-5-(3-methyl-but-2-enyl)-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl]-propionicacid ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 5.17 (t, J=6, 1H), 3.54 (br s,1H), 3.49 (br s, 1H), 3.18-3.37 (m, 2H), 2.70 (t, J=7, 2H), 2.49-2.62(m, 2H), 1.60-2.07 (m, 13H), 1.39-1.48 (m, 1H), 1.05-1.32 (m, 5H).¹³C-NMR (75 MHz CDCl₃) δ (ppm): 180.1, 141.1, 133.9, 133.3, 132.9,122.9, 122.3, 117.4, 73.2, 49.0, 48.9, 40.0, 39.8, 33.6, 32.0, 28.7,26.7, 25.9, 25.6, 23.8, 233.2, 20.5, 18.0. MS ESI-Pos m/z 371.2 (M+H⁺)

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference

1-28. (canceled)
 29. A method of treating a subject suffering fromneurodegenerative or neuroinflammatory disorders, oxidative stressdisorders or mitochondrial disorders, comprising administering to saidsubject a therapeutically effective amount of a compound represented byFormula I:

wherein: -A-B- is —CH₂—(CH₂)₀₋₂—; —CH═CH—; —CH₂—O—; —CH₂—S—; or —CH₂—N—;n is 0 to 5; V is C₇₋₁₂-bicyclo[a.b.c]alkyl;C₇₋₁₂-bicyclo[a.b.c]alkenyl; C₇₋₁₂-heterobicyclo[a.b.c]alkyl; orC₇₋₁₂-heterobicyclo[a.b.c]alkenyl; and a, b, and c are 0 to 6; andwherein the bicyclo ring is optionally substituted with one or moresubstituents selected from C₁₋₆-alkyl, halogen, haloalkyl, carboxy,alkoxycarbonyl, cyano, hydroxy, alkoxy, thiol, and oxo; R¹ is C₁₋₆alkyl; R² is C₁₋₂₀ alkyl; optionally substituted C₂₋₂₀ alkenyl; halogen;hydroxy; alkoxy; acyl; —C(O)OR; —S(O)₂OR; —NR′R″; —NH—C(═NH₂)—NR′R″;—N—SO₂R; —NHC(O)NR′R″; —N(OH)C(O)NR′R″; —SO₂NR′R″; —C(O)NR′R″;—S(O)₀₋₂R′″; —PO(OR)₂; triphenylphosphonium; trialkylphosphonium;optionally substituted aryl; or optionally substituted heterocyclyl; R³is hydrogen; optionally substituted C₁₋₂₀ alkyl; C₂₋₂₀ alkenyl;hydroxyalkyl; acyl; glucoside; phosphoryl; phosphoryloxyalkyl;carboxyalkylcarbonyl; aminoalkylcarbonyl; or alkylketocarbonyl; R⁴ ishydrogen; halogen; nitro, cyano; optionally substituted C₁₋₆alkyl; aryl,arakyl, heterocyclyl or heterocyclylalkyl all optionally substitutedwith alkyl, hydroxy, alkoxy, nitro, acyl, amino, oxo or —C(O)OR;optionally substituted alkenyl; hydroxy, alkoxy, nitro; —C(O)OR;—C(O)NR′R″; —NR′R″; —NHC(O)NR′R″; —NR′—SO₂—R; —NH—C(═NH₂)—NR′R″;—SO₂NR′R″; or —P(O)(OR)₂; or R³ and R⁴ taken together with the atoms towhich they are attached form a heterocyclic ring; R is hydrogen;optionally substituted alkyl; optionally substituted aryl; optionallysubstituted arylalkyl; optionally substituted cycloalkyl; or optionallysubstituted heterocyclyl; R′ and R″ are independently of each otherhydrogen; C₁₋₆ alkyl; hydroxyalkyl; aminoalkyl; optionally substitutedaryl; or optionally substituted benzyl; or R′ and R″ taken together withthe atom to which they are attached form a 5 to 8 membered aromatic,saturated or unsaturated ring, optionally incorporating one additionalatom chosen from N, O, or S and optionally substituted with asubstituent selected from the group consisting of C₁₋₆ alkyl, halo,cyano, alkylthio, lower alkoxy, phenyl, benzyl and carboxy; and R′″ isoptionally substituted C₁₋₆ alkyl; optionally substituted aryl; oroptionally substituted heterocyclyl; or stereoisomers, mixture ofstereoisomers or pharmaceutically acceptable salts thereof.
 30. A methodof treating a subject suffering from neurodegenerative orneuroinflammatory disorders, oxidative stress disorders or mitochondrialdisorders, comprising administering to said subject a therapeuticallyeffective amount of one or more compounds selected from the grouprepresented by the structures:

wherein R is hydrogen or C₁₋₄ alkyl; or stereoisomers, mixture ofstereoisomers or pharmaceutically acceptable salts thereof.
 31. Themethod of claim 29, wherein the subject is suffering from aneurodegenerative disorder selected from stroke, cerebral ischemia,retinal ischemia, post-surgical cognitive dysfunctions, peripheralneuropathy/neuropathic pain, spinal cord injury, head injury andsurgical trauma.
 32. The method of claim 29, wherein the subject issuffering from a mitochondrial disorder selected from epilepsy,Parkinsonism or Parkinson's disease, Alzheimer's disease, amyotrophiclateral sclerosis (ALS), motor neuron diseases, macular degeneration,mitochondrial myopathy, encephalopathy, lactacidosis, stroke (MELAS),myoclonic epilepsy with ragged red fibers (MERFF), Friedreich's ataxiaand cerebellar ataxias.
 33. The method of claim 29, wherein the subjectis suffering from an oxidative stress disorder with inflammatory orautoimmune components selected from diabetes, renal disease,premenstrual syndrome, asthma, chronic obstructive pulmonary disease(COPD), rheumatoid arthritis, osteoarthritis, muscle fatigue, irritablebowel syndrome, inflammatory bowel disease (IBD), premenstrual syndrome(PMS), and intermittent claudication.
 34. The method of claim 29,wherein the subject is suffering from dermatological conditionscharacterized by oxidative stress, selected from age-related skindamage; damage resulting to the skin from insults such as harmfulultraviolet (UV) radiation, pollution, stress and fatigue; contactdermatitis, skin irritation; skin pigmentation; psoriasis; and acne. 35.A method of inhibiting a lipoxygenase enzyme in a subject in need ofsuch inhibition which comprises administering to said subject atherapeutically effective amount of a compound represented by Formula I:

wherein: -A-B- is —CH₂—(CH₂)₀₋₂—; —CH═CH—; —CH₂—O—; —CH₂—S—; or —CH₂—N—;n is 0 to 5; V is C₇₋₁₂-bicyclo[a.b.c]alkyl C₇₋₁₂-bicyclo[a.b.c]alkenyl;C₇₋₁₂-heterobicyclo[a.b.c]alkyl; or C₇₋₁₂-heterobicyclo[a.b.c]alkenyl;and a, b, and c are 0 to 6; and wherein the bicyclo ring is optionallysubstituted with one or more substituents selected from C₁₋₆-alkyl,halogen, haloalkyl, carboxy alkoxycarbonyl, cyano, hydroxy, alkoxy,thiol, and oxo; R¹ is C₁₋₆ alkyl; R² is C₁₋₂₀ alkyl; optionallysubstituted C₂₋₂₀ alkenyl; halogen; hydroxy; alkoxy; acyl; —C(O)OR;—S(O)₂OR; —NR′R″; —NH—C(═NH₂)—NR′R″; —N—SO₂R; —NHC(O)NR′R″;—N(OH)C(O)NR′R″; —SO₂NR′R″; —C(O)NR′R″; —S(O)₀₋₂R′″; —PO(OR)₂;triphenylphosphonium; trialkylphosphonium; optionally substituted aryl;or optionally substituted heterocyclyl; R³ is hydrogen; optionallysubstituted C₁₋₂₀ alkyl; C₂₋₂₀ alkenyl; hydroxyalkyl; acyl; glucoside;phosphoryl; phosphoryloxyalkyl; carboxyalkylcarbonyl;aminoalkylcarbonyl; or alkylketocarbonyl; R⁴ is hydrogen; halogen;nitro; cyano; optionally substituted C₁₋₆alkyl; aryl, arakyl,heterocyclyl or heterocyclylalkyl all optionally substituted with alkyl,hydroxy, alkoxy, nitro, acyl, amino, oxo or —C(O)OR; optionallysubstituted alkenyl; hydroxy; alkoxy; nitro; —C(O)OR; —C(O)NR′R″;—NR′R″; —NHC(O)NR′R″; —NR′—SO₂—R; —NH—C(═NH₂)—NR′R″; —SO₂NR′R″; or—P(O)(OR)₂; or R³ and R⁴ taken together with the atoms to which they areattached form a heterocyclic ring; R is hydrogen; optionally substitutedalkyl; optionally substituted aryl; optionally substituted arylalkyl;optionally substituted cycloalkyl; or optionally substitutedheterocyclyl; R′ and R″ are independently of each other hydrogen; C₁₋₆alkyl; hydroxyalkyl; aminoalkyl; optionally substituted aryl; oroptionally substituted benzyl; or R′ and R″ taken together with the atomto which they are attached form a 5 to 8 membered aromatic, saturated orunsaturated ring, optionally incorporating one additional atom chosenfrom N, O, or S and optionally substituted with a substituent selectedfrom the group consisting of C₁₋₆ alkyl, halo, cyano, alkylthio, loweralkoxy, phenyl, benzyl and carboxy; and R′″ is optionally substitutedC₁₋₆ alkyl; optionally substituted aryl; or optionally substitutedheterocyclyl; or stereoisomers, mixture of stereoisomers orpharmaceutically acceptable salts thereof.
 36. The method of claim 35,wherein the subject is a mammal suffering from asthma or chronicobstructive disease (COPD), arthritis, rheumatoid arthritis,osteoarthritis, allergic rhinitis, psoriasis, atherosclerosis, ordiabetes.
 37. The method of claim 29, wherein the compound is selectedfrom:3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester;3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chroman-2-yl)-propionicacid;2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester;2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicacid dimethyl ester;[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicacid;3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester;4-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexehydro-7,10-methano-2H-benzo[h]chromen-2-yl)-butane-1-sulfonicacid dimethylamide;2-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-(2-Chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;2-Methyl-2-thiazol-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionicacid;2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;4-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-2-methyl-5-propyl-2,4-dihydro-pyrazol-3-one;and single stereoisomers and mixtures of stereoisomers, or thepharmaceutically acceptable salts thereof.
 38. The method of claim 29,wherein the compound is selected from the group represented by thefollowing structures:

or single stereoisomers and mixtures of stereoisomers, or thepharmaceutically acceptable salts thereof.